Riconosciamo Drs. Camillo Ricordi, Antonello Pileggi, R. Damaris Molano, Stephan Speier e Daniel Nyqvist per le discussioni fruttuose. Ringraziamo anche Eleut Hernandez e Diego Espinosa-Heidmann per l&#39;assistenza tecnica, e Mike e Margaret Valdes Formoso aiuto con registrazione video. Byron Maldonado registrare, modificare e prodotto il video finale. Sostegno alla ricerca è stato fornito dal Diabetes Research Institute Foundation ( <a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a> ), il NIH / NIDDK / NIAID (F32DK083226 di MHA, NIH RO3DK075487 ad AC; U01DK089538 a PO.B.). Sostegno alla ricerca addizionale PO.B stato fornito mediante fondi del Karolinska Institutet, il Consiglio svedese della ricerca, la Fondazione Diabete svedese, il Erling Persson-Family Foundation, la Famiglia Knut e Alice Wallenberg Foundation, la Skandia Insurance Company Ltd., VIBRANT ( FP7-228933-2), programma di ricerca strategica nel diabete al Karolinska Institutet, la Novo Nordisk Foundation e Fondazione Berth von Kantzow di.

La seguente procedura viene eseguita sotto la stereoscopio in due fasi, la prima fase consiste nel caricare le isolette nella cannula e la seconda fase è il trapianto effettivo nel ACE. Tutte le procedure eseguite su animali sono stati approvati dalla cura degli animali e del comitato istituzionale uso (IACUC) dell&#39;Università di Miami. 1. Isolotti Caricamento in Cannula per trapianto <ol><li> Centro gli isolotti in piastra di coltura, facendo girare il piatto nei circoli restringimento. </li><li> Scollegare la cannula del &quot;serbatoio&quot; e posizionare la cannula e il tubo di collegamento su una superficie pulita. Il serbatoio può essere realizzata con una punta di 300 ml monouso pipetta di plastica senza filtro (Figura 1a). </li><li> Lavare le bolle d&#39;aria dal serbatoio per garantire flusso continuo di isolette aspirazione quando nel serbatoio. Lavaggio del serbatoio è fatto da portare avanti a mani libere motorizzato siringa-driver usando il pedale(Figura 1b, c). Questo renderà anche spazio nella siringa per consentire l&#39;aspirazione degli isolotti nel serbatoio (pre-caricato con soluzione sterile come supporti salina, PBS o cultura). </li><li> Aspirare delicatamente quantità desiderata di isolotti nel serbatoio. Isolotti tenderà a ricciolo che entrano nel serbatoio e rimarrà insieme verso il basso. L&#39;aspirazione è fatto da guida in retromarcia il motore siringa driver usando il pedale. </li><li> Ricollegare la cannula al serbatoio attraverso il tubo di collegamento. </li><li> Posizionare la punta della cannula indietro nel piatto di coltura e lavare gli isolotti dal serbatoio nel tubo poi nella cannula. Assicurarsi che le isole restare insieme come back-riempire il tubo / cannula delicatamente &quot;sfogliando&quot; (tapping) il tubo (Figura 1d). Arrestare prima o dopo le bolle d&#39;aria prima delle isolette sono lavata la cannula. Se non è sicuro, come smettere isolotti immettere retro della cannula come aria rimanentebolle davanti a isole può aiutare a prevenire il reflusso (riflusso) di isolotti fuori dalla ACE. Will dissipare durante la notte. </li><li> A questo punto, si è pronti a iniettare le isole nel ACE (si prega di vedere i passi successivi). </li></ol> 2. Trapianto di Isole nella camera anteriore dell&#39;occhio <ol><li> Posizionare il mouse anestetizzato su un tappetino caldo sotto stereoscopio. </li><li> Posizionare il muso del mouse in anestesia &quot;maschera&quot; collegato all&#39;ossigeno / macchina di anestesia isoflurano. La maschera è realizzata su una punta di 1 ml pipetta monouso in plastica (senza filtro) e collegata al tubo anestesia attraverso l&#39;estremità stretta (Figura 2a, b). </li><li> Estrarre con cautela le palpebre dell&#39;occhio da trapiantare con il dito indice e il pollice della mano libera e &quot;pop&quot; l&#39;occhio per una migliore esposizione e di facile accesso (Figura 2c). Ciò richiederà una certa pratica per perfezionare senza ostacolare la respirazione del mouse da una pressione eccessiva sulcollo o bloccare il flusso di sangue alla testa. </li><li> Usando una siringa monouso di insulina (29 - 31G) come bisturi, cura solo la punta penetrare nella cornea e fare una singola incisione laterale. Effettuare l&#39;incisione al punto intermedio tra l&#39;apice della cornea e limbus minimizzare riflusso delle isolette durante l&#39;iniezione dal ACE (figura 2d). </li><li> Inserire delicatamente la cannula (precaricato con isolotti) attraverso l&#39;incisione. </li><li> Lentamente espellere isolotti fuori della cannula e depositarli sopra il diaframma. Per evitare il riflusso isolotto causa di un accumulo di pressione eccessiva nel ACE, espellere gli isolotti di spinte brevi come piccolo volume (s) possibile nel quadrante opposto della incisione. Questo può essere ulteriormente garantita da compattazione isolotti nel tubo durante il caricamento della cannula (si prega di vedere il punto 1.6). </li><li> Lentamente ritirare la cannula dalla ACE. Questa è una fase critica, in particolare, se un grande volume di isolotti stato iniettato come riflusso isolotto causa presche accumula all&#39;interno del ACE può essere inevitabile. Per eliminare / ridurre al minimo il reflusso isolotto, ruotare delicatamente la cannula mentre all&#39;interno della ACE per scaricare la pressione in eccesso attraverso l&#39;incisione intorno alla cannula. Verificare la presenza di segni di reflusso come si tenta di ritrarre la cannula e, se necessario, attendere fino alla scomparsa di pressione prima di ritrarre completamente la cannula dalla ACE. </li><li> Sciacquare l&#39;occhio trapiantato con PBS sterile o soluzione fisiologica. </li><li> Iniettare buprenorfina per postoperatoria (0,05-0,1 mg / kg, per via sottocutanea) per le prime 48 ore. </li><li> Applicare oftalmica unguento antibiotico eritromicina a occhio trapiantato immediatamente dopo il trapianto. </li><li> Posizionare l&#39;animale indietro in una gabbia riscaldata per permettere il recupero dall&#39;anestesia. </li></ol>

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PO.B. è uno dei fondatori della Biocrine società biotech, che sta per utilizzare la camera anteriore dell&#39;occhio come una piattaforma di servizio commerciale. AC è il brevetto che protegge questa tecnologia.

Isole pancreatiche murine sono state isolate mediante digestione con collagenasi seguita da purificazione su gradienti di densità, come descritto in precedenza 33. Isole isolate sono state coltivate durante la notte prima del trapianto. Mentre questo può non essere necessario, si raccomanda di consentire gli isolotti di recuperare dalla procedura di isolamento. Questo è fondamentale quando il trapianto viene eseguito nei pazienti diabetici in quanto garantirà il trapianto di sopravvivere / isole robuste. ...

I progressi nella microscopia intravitale hanno rivelato fenomeni fisiologici non previsto da studi in vitro 1. Questo mette in evidenza la sfida di tradurre i risultati ottenuti con convenzionali metodi in vitro nell&#39;animale vivente. Negli ultimi dieci anni, la visualizzazione dei tessuti negli animali vivi è stata notevolmente migliorata dai progressi tecnologici in modalità di imaging 2, 3, 4, 5, 6. Questo ha stimolato la necessità di approcci di imaging in vivo con applicazione realizzabile in modelli animali sperimentali per consentire la visualizzazione longitudinale dei tessuti bersaglio non invasivo. Le tecniche di imaging come la risonanza magnetica e la tomografia ad emissione di positroni o bioluminescenza hanno permesso non invasiva per immagini di organi / tessuti in profondità all&#39;interno del corpo 7-8, 9. Ma queste tecniche non possono realizzare singola cella di risoluzione a causa di segnali di fondo e alla bassa risoluzione spaziale, nonostante l&#39;uso of materiali ad alto contrasto o di tessuto-specifica 4 luminescenza. Questo è stato affrontato con l&#39;avvento di due fotoni microscopia a fluorescenza confocale 10. Due-fotone microscopia abilitato studi di imaging intravitale per visualizzare e quantificare gli eventi cellulari con dettagli senza precedenti 11, 12. Questo ha portato alla caratterizzazione dei principali processi biologici in salute e malattia 13, 14, 15, 16. Mentre pionieristici studi di imaging intravitale sono principalmente &quot;imitato&quot; in condizioni in vivo nel tessuto asportato (linfonodi ad esempio), altri studi hanno utilizzato poco invasive per tessuti bersaglio di immagini esposte in situ 17, 18, ​​19, 20, 21. Altri studi hanno utilizzato anche modelli a camera &quot;finestra&quot; per aggirare le limitazioni connesse con approcci invasivi e la risoluzione limitata di imaging in vivo 22, 23, 24, 25. Nel modello camera di finestra, una camera con una finestra trasparente viene impiantato chirurgicamente nella pelle con diffelocalità affitto (pelle dorsale o l&#39;orecchio, mammaria cuscinetto adiposo, fegato, ecc) su l&#39;animale (ad esempio topo, ratto, coniglio). Anche se questo approccio consente in modo chiaro ad alta risoluzione di immagini in vivo, che richiede un intervento chirurgico invasivo per impiantare la camera e non può essere in grado di ospitare studi di imaging longitudinale su diverse settimane o mesi 22. È stato recentemente dimostrato che la combinazione di alta risoluzione microscopia confocale con una procedura minimamente invasiva, ossia trapianto nella camera anteriore dell&#39;occhio (ACE) fornisce una &quot;finestra naturale corpo&quot; come un potente e versatile in vivo imaging piattaforma 26, 27. Trapianto nel ACE è stato usato negli ultimi decenni per studiare aspetti biologici di una varietà di tessuti 28, 29, 30, e la sua combinazione con la recente imaging ad alta risoluzione abilitato studiando la fisiologia delle isole pancreatiche con singola cella di risoluzione non- invasiva e longitudinalmente &lt;sup&gt; 26, 27. Questo approccio è stato utilizzato per studiare le risposte autoimmuni durante lo sviluppo di diabete di tipo 1 in modelli animali (dati non pubblicati). E &#39;stato anche utilizzato per studiare lo sviluppo del pancreas, così come, negli studi di funzione renale da trapianto in gemme pancreatiche l&#39;asso o singoli glomeruli renali, rispettivamente (dati non pubblicati). Un recente rapporto di questo approccio ulteriormente dimostrato la sua applicazione per studiare la risposta immunitaria dopo trapianto di isole pancreatiche 31. Importante, questo studio ha dimostrato che il trapianto nella camera anteriore dell&#39;occhio fornisce una finestra naturale del corpo per eseguire: (1) longitudinale, non invasiva dei tessuti trapiantati in vivo; (2) in vivo per valutare cytolabeling fenotipo cellulare e la vitalità in situ, (3) monitoraggio in tempo reale di infiltrare cellule immunitarie nel tessuto bersaglio, e (4) intervento locale per l&#39;applicazione topica o iniezione intraoculare. Qui, Siamo demonstrate come eseguire il trapianto nella camera anteriore dell&#39;occhio utilizzando isole pancreatiche.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Descrizione / Commenti </td></tr><tr><td> IsoTHESIA (isoflurano) </td><td> Buttler Animal Health di alimentazione </td><td> 11695-6775-2 </td><td> 99,9% isoflurano / ml </td></tr><tr><td> Ketaset (ketamina HCL) </td><td> Fort Dodge Animal Health </td><td> 0856-2013-01 </td><td> Alternative anestesia iniettabile </td></tr><tr><td> Beprenex (buprenorfina 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> Eritromicina unguento oftalmico USP, 0,5% </td><td> Akron </td><td> 17478-070-35 </td><td> Applicato profilassi per gli occhi trapiantati </td></tr><tr><td> Di sodio cloruro 0,9% (Saline) </td><td> Hospira Inc. </td><td> 0409-7983-03 </td><td> Per iniezione endovenosa. Sterile </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td> 1X. Sterile </td></tr><tr><td> CMRL media 1066 </td><td> Cellgro </td><td> 98-304-CV </td><td> Completato, CIT modifica. Mezzi preferiti per isole </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 immagini è emerso come uno strumento indispensabile nella ricerca biologica. Nel processo, tecniche di imaging sono state sviluppate molte differenti per studiare processi biologici negli animali non invasivo. Tuttavia, un grande limite tecnico in vigore le modalità di imaging intravitale è l&#39;incapacità di combinare non invasivo, l&#39;imaging longitudinale con cella singola capacità di risoluzione. Mostriamo qui come il trapianto nella camera anteriore dell&#39;occhio elude tale limitazione significativa che offre una piattaforma versatile sperimentale che permette di non invasivo, l&#39;imaging longitudinale con risoluzione cellulare in vivo. Dimostriamo la procedura di trapianto nel topo e di fornire risultati rappresentativi utilizzando un modello con rilevanza clinica, in particolare trapianto di isole pancreatiche. Oltre a consentire la visualizzazione diretta in una varietà di tessuti trapiantati nella camera anteriore dell&#39;occhio, questo approccio fornisce una piattaforma per ghialonefarmaci n eseguendo a lungo termine di follow-up e il monitoraggio nei tessuti bersaglio. Grazie alla sua versatilità, tessuto / trapianto di cellule nella camera anteriore dell&#39;occhio non solo terapie di trapianto benefici, si estende ad altre applicazioni in vivo per studiare processi fisiologici e fisiopatologici quali trasduzione del segnale e nel cancro o sviluppo della malattia autoimmune.

Ci sono alcuni parametri che definiscono un trapianto &quot;buono&quot;. Un trapianto buona è quella che procede senza sanguinamento quando si effettua l&#39;incisione come si può vedere nel video. Sanguinamento è impedito / minimizzato penetrare solo la punta del bisturi (aghi) nel ACE (Figura 3a). Questo aiuterà anche a prevenire il contatto e la puntura dell&#39;iride. Essa garantirà inoltre una piccola incisione che guarirà molto bene senza causare opacità della cornea nel tempo (figu...

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

Un nuovo approccio che combina il trapianto intraoculare e la microscopia confocale permette longitudinale, non invasivo imaging in tempo reale con singola cella di risoluzione all&#39;interno di tessuti innestati<em&gt; In vivo</em&gt;. Dimostriamo come trapiantare isole pancreatiche nella camera anteriore dell&#39;occhio mouse.

Trapianto nella camera anteriore dell&#39;occhio per longitudinale, non invasiva<em&gt; In vivo</em&gt; Imaging con Single-cella di risoluzione in tempo reale

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 Views.  University of Miami Miller School of Medicine. Un nuovo approccio che combina il trapianto intraoculare e la microscopia confocale permette longitudinale, non invasivo imaging in tempo reale con singola cella di risoluzione all'interno di tessuti innestati In vivo. Dimostriamo come trapiantare isole pancreatiche nella camera anteriore dell'occhio mouse.

Video: 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.

Ad alta frequenza ad alta risoluzione Ecocardiografia: Prima Prova a non invasiva misura ripetuta di deformazione miocardica, contrattilità, e rigurgito mitralico in Ischemia-riperfuso Cuore Murine

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

Ricerca

Medicina

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.

Multispettrali in tempo reale immagini a fluorescenza per il rilevamento intraoperatoria del linfonodo sentinella in Oncologia Ginecologica

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.

Imaging non invasivo di acuta Alloinnesto rigetto dopo trapianto renale Rat Utilizzo<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.

Sviluppo di un audio-based ambiente di gioco virtuale per assistere con doti di navigazione 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 ...

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.

Chemioterapia-indotta vascolare Tossicità - in tempo reale<em&gt; In vivo</em&gt; Imaging di 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 ...

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.

Valutazione non invasiva della efficacia di nuove terapie per patologie intestinali Utilizzando Serial Imaging endoscopica Live Mice

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.

Incapsulamento termogenico preadipocytes al trapianto nel tessuto adiposo Depositi tessuti

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.

Esteso Time-lapse Imaging intravitale del Real-time multicellulare Dynamics nel microambiente tumorale

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.

Longitudinale<em&gt; In Vivo</em&gt; Imaging del Cerebrovasculature: Rilevanza per le malattie del sistema nervoso centrale

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.

Ri-Arterialized trapianto parziale di fegato del ratto con un in vivo vaso-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. ...