Nous reconnaissons les Drs. Camillo Ricordi, Antonello Pileggi, R. Damaris Molano, Stephan Speier et Daniel Nyqvist pour des discussions fructueuses. Nous remercions également Eleut Hernandez et Diego Espinosa-Heidmann pour l&#39;assistance technique, et Mike Valdes et Margaret Formoso de l&#39;aide pour l&#39;enregistrement vidéo. Byron Maldonado a enregistré, édité et produit la vidéo finale. Soutien à la recherche a été fourni par la Fondation Diabetes Research Institute ( <a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a> ), le NIH / NIDDK / NIAID (F32DK083226 à MHA; NIH RO3DK075487 à AC; U01DK089538 à PO.B.). Soutien additionnel à la recherche PO.B a été fournie par des fonds provenant du Karolinska Institutet, le Conseil de recherche suédois, la Fondation du diabète suédois, la famille Erling Persson-Foundation, la famille Knut et Alice Wallenberg Foundation, le Skandia Insurance Company Ltd, VIBRANT ( FP7-228933-2), le Programme stratégique de recherche sur le diabète au Karolinska institutet, la Fondation Novo Nordisk, et la Fondation Berth von Kantzow l&#39;.

La procédure suivante est réalisée dans le stéréoscope en 2 étapes, la première étape consiste à charger les îlots dans la canule et la seconde étape est la transplantation réelle dans l&#39;ACE. Toutes les procédures effectuées sur des animaux ont été approuvés par les soins des animaux des établissements et des comités utilisation (IACUC) de l&#39;Université de Miami. 1. Chargement en îlots Canule destinés à la transplantation <ol><li> Centre des îlots en boîte de culture en faisant tourner le plat dans les milieux rétrécissement. </li><li> Débrancher le canule du «réservoir» et placer la canule et tubulure de raccordement sur une surface propre. Le réservoir peut être faite de pointe de 300 ul pipette jetable en plastique sans filtre (figure 1a). </li><li> Rincer les bulles d&#39;air hors du réservoir pour assurer flux continu d&#39;îlots lors de l&#39;aspiration dans le réservoir. Rinçage du réservoir se fait en faisant avancer le mains-libres motorisée pousse-seringue à l&#39;aide de la pédale(Figure 1b, c). Cela permettra également de libérer de l&#39;espace dans la seringue pour permettre l&#39;aspiration des îlots dans le réservoir (pré-chargé avec une solution stérile de sérum physiologique tels que les médias, la culture ou PBS). </li><li> Aspirer la quantité désirée d&#39;îlots dans le réservoir. Îlots aura tendance à tourbillonner comme ils entrer dans le réservoir et restera ensemble vers le bas. L&#39;aspiration se fait par l&#39;arrière du volant moteur pousse-seringue à l&#39;aide de la pédale. </li><li> Reconnecter la canule dans le réservoir par l&#39;intermédiaire de la tubulure de raccordement. </li><li> Placer la pointe de la canule de retour dans la boîte de culture et la vidange des îlots sur le réservoir dans le tube puis dans la canule. Assurez-vous que les îlots restent ensemble que vous sauvegardez-remplir la tubulure / canule en douceur &quot;pichenette&quot; (taraudage) du tube (figure 1d). Arrêter, avant ou après que toutes les bulles d&#39;air avant de les îlots sont évacués de la canule. En cas de doute, arrêtez-vous îlots entre l&#39;arrière de la canule que l&#39;air restebulles qui viennent d&#39;îlots peut aider à prévenir le reflux (reflux) des îlots de l&#39;ACE. Permettra de dissiper la nuit. </li><li> A ce stade, vous êtes prêt à injecter des îlots dans le ACE (s&#39;il vous plaît voir les prochaines étapes). </li></ol> 2. Transplantation d&#39;îlots dans la chambre antérieure de l&#39;oeil <ol><li> Positionnez la souris anesthésiée sur un tapis chaud sous stéréoscope. </li><li> Placez le museau de la souris dans l&#39;anesthésie &quot;masque&quot; connecté à l&#39;oxygène / machine anesthésie à l&#39;isoflurane. Le masque est réalisé en une matière plastique de 1 ml embout de pipette jetable (sans filtre) et relié à un tube d&#39;anesthésie par l&#39;extrémité étroite (Figure 2a, b). </li><li> Doucement rétracter les paupières de l&#39;œil pour être transplantés avec l&#39;index et le pouce de votre main libre et &quot;pop&quot; l&#39;oeil pour une meilleure exposition et un accès facile (figure 2c). Cela nécessitera un peu de pratique pour perfectionner sans gêner la respiration de la souris par une pression excessive sur lecou ou bloquer l&#39;écoulement du sang à la tête. </li><li> En utilisant une seringue à insuline jetable (29 - 31G) comme un scalpel, bien que la pointe pénètre dans la cornée et faire une incision latérale unique. Réaliser l&#39;incision à mi-chemin entre le sommet de la cornée et du limbe de minimiser le reflux des îlots lors de l&#39;injection sur l&#39;ACE (figure 2d). </li><li> Soigneusement insérer la canule (préchargé avec îlots) à travers l&#39;incision. </li><li> Lentement éjecter îlots sur la canule et les déposer sur le dessus de l&#39;iris. Pour éviter le reflux îlot en raison de l&#39;accumulation de pression excessive dans l&#39;ACE, éjectez les îlots de coups brefs que peu de volume (s) possible dans le quadrant opposé à l&#39;incision. Cela peut être également assurée par compactage îlots dans le tube pendant le chargement de la canule (s&#39;il vous plaît voir l&#39;étape 1.6). </li><li> Lentement retirer la canule de l&#39;ACE. Il s&#39;agit d&#39;une étape cruciale, en particulier, si un grand nombre d&#39;îlots a été injecté sous forme de reflux îlot en raison de pressionsque s&#39;accumuler à l&#39;intérieur de l&#39;ACE peut-être inévitable. Pour éliminer / réduire les reflux îlot, faites tourner doucement la canule tout l&#39;intérieur de l&#39;ACE pour relâcher la pression en excès à travers l&#39;incision autour de la canule. Vérifiez les signes de reflux que vous essayez de retirer la canule et, si nécessaire, attendez jusqu&#39;à la disparition de la pression avant de se rétracter complètement la canule de l&#39;ACE. </li><li> Rincer l&#39;œil greffé avec du PBS stérile ou une solution saline. </li><li> Injecter de la buprénorphine pour l&#39;analgésie postopératoire (0,05-0,1 mg / kg, voie sous-cutanée) pour les 48 premières heures. </li><li> Appliquer une pommade ophtalmique antibiotique érythromycine à l&#39;oeil transplanté immédiatement après la transplantation. </li><li> Placez l&#39;animal dans une cage arrière réchauffé pour permettre la récupération de l&#39;anesthésie. </li></ol>

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PO.B. est l&#39;un des fondateurs de la société de biotechnologie Biocrine, qui va utiliser la chambre antérieure de l&#39;œil comme une plate-forme de service commercial. AC est sur le brevet protégeant cette technologie.

Îlots pancréatiques murins ont été isolés par digestion à la collagénase suivie d&#39;une purification sur gradient de densité, comme décrit précédemment 33. Îlots isolés ont été cultivées pendant une nuit avant la transplantation. Bien que cela ne soit pas nécessaire, il est recommandé de permettre aux îlots de se remettre de la procédure d&#39;isolement. Cela est essentiel si la transplantation est effectuée chez les receveurs diabétiques car elle garantit la transplantation d&#39;îlo...

Les progrès de la microscopie intravitale ont révélé des phénomènes physiologiques non prévus par les études in vitro 1. Cela met en évidence le défi de traduire les résultats obtenus par des méthodes in vitro conventionnelle dans l&#39;animal vivant. Dans la dernière décennie, la visualisation des tissus chez les animaux vivants a été considérablement améliorée par les avancées technologiques en 2 modalités d&#39;imagerie, 3, 4, 5, 6. Cela a suscité un besoin pour des approches d&#39;imagerie in vivo avec une application possible dans des modèles animaux expérimentaux pour permettre la visualisation longitudinale des tissus cibles de façon non invasive. Les techniques d&#39;imagerie telles que l&#39;imagerie par résonance magnétique et la tomographie par émission de positons ou bioluminescence ont permis à l&#39;imagerie non invasive des organes / tissus profonds dans le corps 7-8, 9. Mais ces techniques ne peuvent pas réaliser seule cellule de résolution en raison de signaux de fond élevés et la faible résolution spatiale, en dépit de l&#39;utilisation of matériaux à fort contraste ou tissu-spécifique 4 luminescence. Cela a été abordée avec l&#39;avènement de la microscopie à deux photons de fluorescence confocale 10. Microscopie à deux photons a permis des études d&#39;imagerie intravitale de visualiser et de quantifier les événements cellulaires avec des détails sans précédent 11, 12. Cela a conduit à la caractérisation des processus biologiques essentiels en matière de santé et de la maladie 13, 14, 15, 16. Alors que pionnier des études d&#39;imagerie ont principalement intravitale &quot;imité&quot; in vivo dans le tissu excisé (ganglions lymphatiques, par exemple), d&#39;autres études ont utilisé des approches invasives dans les tissus cibles d&#39;images exposées in situ 17, 18, ​​19, 20, 21. D&#39;autres études ont également utilisé des &quot;modèles&quot; fenêtre de la chambre de contourner les limitations associées à des approches invasives et la résolution d&#39;imagerie in vivo limitée 22, 23, 24, 25. Dans le modèle de fenêtre de chambre, une chambre avec une fenêtre transparente est implanté chirurgicalement sous la peau à difféemplacements de loyer (peau du dos ou de l&#39;oreille, mammaire coussinet adipeux, le foie, etc) sur l&#39;animal (par exemple souris, rat, lapin). Bien que cette approche permet clairement à haute résolution pour l&#39;imagerie in vivo, il nécessite une chirurgie invasive pour implanter la chambre et peut ne pas être en mesure d&#39;accueillir les études d&#39;imagerie longitudinales sur plusieurs semaines ou mois 22. Il a été récemment démontré que la combinaison de haute résolution microscopie confocale à une procédure minimalement invasive, à savoir la transplantation dans la chambre antérieure de l&#39;œil (ACE) offre une «fenêtre naturelle du corps&quot; comme un. Puissant et polyvalent d&#39;imagerie in vivo plate-forme 26, 27 Transplantation dans le ACE a été utilisé dans les dernières décennies pour étudier les aspects biologiques d&#39;une grande variété de tissus 28, 29, 30, et sa combinaison récente avec l&#39;imagerie haute résolution a permis l&#39;étude de la physiologie des îlots pancréatiques avec une seule cellule de résolution non- invasive et longitudinalement &lt;sup&gt; 26, 27. Cette approche a été utilisée pour étudier les réactions auto-immunes au cours du développement du diabète de type 1 dans des modèles animaux (données non publiées). Il a également été utilisé pour étudier le développement du pancréas, ainsi que, dans les études de la fonction rénale par la transplantation dans les bourgeons pancréatiques de l&#39;ECA ou individuels glomérules rénaux, respectivement (données non publiées). Un rapport récent de cette approche en outre démontré son application à étudier les réponses immunitaires après transplantation d&#39;îlots pancréatiques 31. Surtout, cette étude a montré que la transplantation dans la chambre antérieure de l&#39;oeil offre une fenêtre naturelle du corps pour effectuer: (1) longitudinal, l&#39;imagerie non-invasive de tissus transplantés in vivo, (2) in vivo pour évaluer cytolabeling phénotype cellulaire et la viabilité de situ, (3) suivi en temps réel de l&#39;infiltration des cellules immunitaires dans le tissu cible, et (4) une intervention locale en application topique ou par injection intra-oculaire. Ici, nous avons Demonstrate comment effectuer la transplantation dans la chambre antérieure de l&#39;oeil à l&#39;aide des îlots pancréatiques.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Description / Commentaires </td></tr><tr><td> IsoTHESIA (isoflurane) </td><td> Alimentation Buttler la santé animale </td><td> 11695-6775-2 </td><td> 99,9% isoflurane / ml </td></tr><tr><td> Ketaset (kétamine HCL) </td><td> Fort Dodge Animal Health </td><td> 0856-2013-01 </td><td> Alternative anesthésie injectable </td></tr><tr><td> Beprenex (buprénorphine HCL) </td><td> Reckitt Benckiser Health Care (UK) Ltd </td><td> 12496-075-7-1 </td><td> 0,3 mg / ml </td></tr><tr><td> USP érythromycine onguent ophtalmique, 0,5% </td><td> Akron </td><td> 17478-070-35 </td><td> Appliqué à titre prophylactique pour les yeux transplanté </td></tr><tr><td> Chlorure de sodium à 0,9% (Saline) </td><td> Hospira Inc </td><td> 0409-7983-03 </td><td> Pour injection intraveineuse. Stérile </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td> 1X. Stérile </td></tr><tr><td> CMRL moyenne 1066 </td><td> Cellgro </td><td> 98-304 CV- </td><td> Complétée, la modification CIT. Support privilégié pour les îlots </td></tr></tbody></table>

transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time

Intravitale imagerie est devenue un outil indispensable dans la recherche biologique. Dans le processus, les techniques d&#39;imagerie nombreux ont été développés pour étudier différents processus biologiques chez les animaux non-invasive. Cependant, une limitation technique majeur existants modalités d&#39;imagerie intravitale est l&#39;incapacité de combiner non invasive, l&#39;imagerie longitudinale avec des capacités de résolution unicellulaires. Nous montrons ici comment la transplantation dans la chambre antérieure de l&#39;œil contourne cette limitation significative en offrant une plate-forme expérimentale polyvalente qui permet aux non-invasive, l&#39;imagerie longitudinale avec une résolution cellulaire in vivo. Nous démontrons la procédure de transplantation chez la souris et de fournir des résultats représentatifs à l&#39;aide d&#39;un modèle avec pertinence clinique, à savoir transplantation d&#39;îlots pancréatiques. En plus de permettre une visualisation directe dans une variété de tissus transplantés dans la chambre antérieure de l&#39;œil, cette approche fournit une plate-forme d&#39;éboulisn en effectuant des médicaments à long terme de suivi et de surveillance dans les tissus cibles. En raison de sa polyvalence, le tissu / la transplantation de cellules dans la chambre antérieure de l&#39;œil non seulement les thérapies de transplantation avantages, elle s&#39;étend à d&#39;autres applications in vivo pour étudier les processus physiologiques et physiopathologiques tels que la transduction du signal et de cancers ou développer maladie auto-immune.

Il ya quelques paramètres qui définissent une «bonne» la transplantation. Une transplantation bonne est celle qui se déroule sans saignement lors de l&#39;incision comme on peut le voir dans la vidéo. Le saignement est empêché / minimisée en pénétrant que la pointe du scalpel (aiguille) dans le ACE (figure 3a). Cela aidera également à éviter tout contact et à la perforation de l&#39;iris. Il assurera également une petite incision qui va guérir très bien sans causer de trouble de la cor...

Watch this Scientific Journal Video about Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time at JoVE.com

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Une nouvelle approche combinant la greffe intraoculaire et la microscopie confocale permet longitudinale, non-invasive imagerie en temps réel avec une seule cellule de résolution au sein des tissus greffés<em&gt; In vivo</em&gt;. Nous démontrons comment transplanter les îlots pancréatiques dans la chambre antérieure de l&#39;œil de la souris.

Transplantation dans la chambre antérieure de l&#39;oeil pour longitudinale, non-invasive<em&gt; In vivo</em&gt; Imagerie avec une seule cellule de résolution en temps réel

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

transplantation-into-anterior-chamber-eye-for-longitudinal-non

Research

JoVE Journal

Medicine

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

13.3K vues.  University of Miami Miller School of Medicine. Une nouvelle approche combinant la greffe intraoculaire et la microscopie confocale permet longitudinale, non-invasive imagerie en temps réel avec une seule cellule de résolution au sein des tissus greffés In vivo. Nous démontrons comment transplanter les îlots pancréatiques dans la chambre antérieure de l'œil de la souris.

Vidéo: Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Haute-fréquence de haute résolution échocardiographie: première preuve de non-invasive mesure répétée de la souche du myocarde, la contractilité, et la régurgitation mitrale au coeur d&#39;ischémie-reperfusion murin

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

Recherche

Médecine

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Multispectrale en temps réel d&#39;imagerie par fluorescence pour la détection peropératoire du ganglion sentinelle en oncologie gynécologique

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

L&#39;imagerie non-invasive de rejet aigu après transplantation rénale Rat aide<sup&gt; 18</sup&gt; F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Développement d&#39;un environnement Audio-based jeux virtuels pour aider avec des compétences de navigation dans les aveugles

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Induits par la chimiothérapie vasculaire Toxicité - en temps réel<em&gt; In vivo</em&gt; Imagerie de déficience navire

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Évaluation non invasive de l&#39;efficacité de nouveaux traitements pour les pathologies intestinales en utilisant l&#39;imagerie endoscopique de série de souris vivantes

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Encapsulation thermogénique préadipocytes pour la transplantation dans dépôts de tissu adipeux

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Time-lapse Extended Imagerie intravitale du temps réel multicellulaire Dynamics dans le microenvironnement de la tumeur

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Longitudinal<em&gt; In Vivo</em&gt; Imagerie du Cerebrovasculature: Pertinence pour les maladies du système nerveux central

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Re-artérialisé Transplantation de foie partielle de Rat in vivo axée sur le navire 70 % hépatectomie

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...