Reconhecemos os drs. Camillo Ricordi, Antonello Pileggi, R. Damaris Molano, Stephan Speier e Daniel Nyqvist para discussões frutíferas. Agradecemos também Eleut Hernandez e Diego Espinosa-Heidmann para assistência técnica, e Mike Valdes e Margaret Formoso ajuda com gravação de vídeo. Byron Maldonado gravados, editados e produziu o vídeo final. Apoio à pesquisa foi fornecido pelo Instituto de Pesquisa do Diabetes Foundation ( <a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a> ), o NIH / NIDDK / NIAID (F32DK083226 a MHA; NIH RO3DK075487 a AC; U01DK089538 para PO.B.). Apoio à pesquisa adicional para PO.B foi fornecido através de fundos do Karolinska Institutet, da Suécia Research Council, a Fundação Diabetes sueca, a Família Erling Persson-Fundação, a Família Knut e Alice Wallenberg Foundation, a Skandia Insurance Company Ltd., VIBRANTE ( FP7-228933-2), Programa de Pesquisa Estratégica em Diabetes no Karolinska Institutet, a Novo Nordisk Foundation e Fundação Berth von Kantzow de.

O procedimento seguinte é realizado sob a estereoscópio em 2 passos, o primeiro passo envolve o carregamento das ilhotas para dentro da cânula e a segunda etapa é o transplante de real no ACE. Todos os procedimentos realizados em animais foram aprovados pelo cuidado com os animais institucional e comissão de uso (IACUC), da Universidade de Miami. 1. Ilhotas de carga em cânula para Transplante <ol><li> Centro das ilhotas no prato de cultura girando o prato em círculos mais estreitos. </li><li> Desligar a cânula da &quot;reservatório&quot; e colocar a cânula eo tubo de ligação sobre uma superfície limpa. O reservatório pode ser feito de uma ponta de pipeta de plástico descartável 300 ul sem filtro (Figura 1a). </li><li> Lave as bolhas de ar para fora do reservatório para assegurar fluxo contínuo de ilhotas quando a aspiração no reservatório. A lavagem do reservatório é feito por impulsionar o mãos-livres motorizada seringa motorista usando o pedal(Figura 1b, c). Isto também irá criar espaço dentro da seringa para permitir que a aspiração das ilhotas para o reservatório (pré-carregado com uma solução estéril tal como a media salina, PBS ou cultura). </li><li> Aspirar cuidadosamente quantidade desejada de ilhotas para o reservatório. Ilhotas tenderá a rodar à medida que entram no reservatório e permanecerá em conjunto em direcção ao fundo. A aspiração é feita por condução motorizada para trás a seringa motorista usando o pedal. </li><li> Reconecte a cânula para o reservatório através da tubagem de ligação. </li><li> Colocar a ponta da cânula para trás na placa de cultura e lavar as ilhotas para fora do reservatório para dentro do tubo, em seguida, para dentro da cânula. Certifique-se de que as ilhotas permanecer juntos como de volta a encher o tubo / cânula suavemente &quot;sacudir&quot; (derivação) do tubo (Figura 1d). Parar, antes ou depois de todas as bolhas de ar antes de as ilhotas são lavadas para fora da cânula. Se não for claro, parar como ilhotas entrar na parte de trás da cânula enquanto o ar restantebolhas antes de ilhotas pode ajudar a prevenir o refluxo (retorno) de ilhotas fora da ACE. Dissipará durante a noite. </li><li> Nesta fase, você está pronto para injetar as ilhotas na ACE (por favor, consulte os passos seguintes). </li></ol> 2. Islet Transplantation dentro da câmara anterior do olho <ol><li> Posicione o mouse anestesiados em uma almofada quente sob estereoscópio. </li><li> Coloque o focinho do mouse em &quot;máscara&quot; anestesia ligado ao oxigênio / máquina de anestesia isoflurano. A máscara é feita de uma ponta de plástico descartável 1 ml pipeta (sem filtro), e ligada ao tubo da anestesia através da parte mais estreita (Figura 2a, b). </li><li> Gentilmente retrair as pálpebras do olho a ser transplantado utilizando o dedo indicador eo polegar de sua mão livre e &quot;pop&quot; do olho para fora para melhor exposição e fácil acesso (Figura 2c). Isso vai exigir um pouco de prática para aperfeiçoar sem impedindo a respiração do mouse por uma pressão excessiva sobre opescoço ou bloqueando o fluxo de sangue para a cabeça. </li><li> Utilizando uma seringa descartável de insulina (29 - 31G) como bisturi, com cuidado apenas a ponta penetrar na córnea e fazer uma única incisão lateral. Fazer a incisão no ponto médio entre o vértice da córnea e limbo para minimizar o refluxo dos ilhéus durante a injecção para fora do ACE (figura 2d). </li><li> Cuidadosamente inserir a cânula (pré-carregado com ilhotas) através da incisão. </li><li> Lentamente ejectar ilhotas para fora da cânula e depositá-los sobre a íris. Para evitar o refluxo de ilhotas devido à acumulação de pressão excessiva no ACE, ejectar as ilhotas de impulsos breves como pequeno volume (s) possível no quadrante oposto ao da incisão. Isto pode ser ainda assegurada por compactação ilhotas na tubagem durante o carregamento da cânula (por favor ver passo 1.6). </li><li> Lentamente retrair a cânula para fora da ACE. Este é um passo crítico, especialmente, se um grande volume de ilhotas foi injectado como refluxo islet devido à presse construir dentro da ACE pode ser inevitável. Para eliminar / minimizar refluxo ilhota, gire gentilmente a cânula enquanto dentro da ACE para liberar o excesso de pressão através da incisão ao redor da cânula. Verifique se há sinais de refluxo como você tentar retirar a cânula e, se necessário, aguarde até que desapareça completamente a pressão antes de retração da cânula para fora da ACE. </li><li> Lavar o olho transplantado com PBS estéril ou soro fisiológico. </li><li> Injectar buprenorfina para analgesia pós-operatória (0,05-0,1 mg / kg, por via subcutânea) a 48 h em primeiro lugar. </li><li> Aplicar pomada oftálmica antibiótico eritromicina ao olho transplantado imediatamente após o transplante. </li><li> Colocar o animal numa gaiola de volta aquecido para permitir a recuperação da anestesia. </li></ol>

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PO.B. é um dos fundadores da empresa de biotecnologia Biocrine, que vai usar a câmara anterior do olho como uma plataforma de serviços comerciais. AC é sobre a patente proteger esta tecnologia.

Ilhéus pancreáticos de murino foram isolados usando a digestão com colagenase seguido por purificação em gradiente de densidade, tal como descrito previamente 33. Ilhotas isoladas foram cultivadas durante a noite antes do transplante. Embora isto possa não ser necessário, é recomendado para permitir que as ilhotas para recuperar do procedimento de isolamento. Isto é crítico, quando é realizado o transplante em receptores diabéticos uma vez que irá assegurar o transplante de ilhéus sobreviventes ...

Os avanços na microscopia intravital revelaram fenómenos fisiológicos não preditos por estudos in vitro 1. Isto realça o desafio na tradução resultados obtidos por métodos in vitro convencional para o animal vivo. Na última década, a visualização de tecidos em animais vivos foi consideravelmente melhorada por avanços tecnológicos em modalidades de imagem 2, 3, 4, 5, 6. Isto estimulou a necessidade de abordagens de imagiologia in vivo, com aplicação possível em modelos animais experimentais, para permitir a visualização de tecidos-alvo longitudinal de forma não invasiva. Técnicas de imagem, como ressonância magnética e tomografia por emissão de pósitrons ou bioluminescência permitiram não-invasivas de órgãos / tecidos profundos dentro do corpo 7-8, 9. Mas estas técnicas não podem atingir única célula de resolução devido aos sinais de fundo elevados e de resolução espacial baixa, a despeito do uso of materiais de elevado contraste ou tecido específico 4 luminescência. Esta foi tratada com o advento de dois fotões microscopia de fluorescência confocal 10. Dois fótons de microscopia intravital habilitado estudos de imagem para visualizar e quantificar eventos celulares com detalhes sem precedentes 11, 12. Isto conduziu à caracterização dos principais processos biológicos na saúde e na doença de 13, 14, 15, 16. Embora os estudos de imagem pioneiras intravital têm essencialmente &quot;imitada&quot; em condições in vivo em tecido excisado (por exemplo, nódulos linfáticos), outros estudos usaram abordagens invasivas em tecidos-alvo de imagem expostos in situ 17, 18, ​​19, 20, 21. Outros estudos também têm utilizado &quot;modelos de janelas de câmara&quot; para contornar limitações associadas a abordagens invasivas e resolução de imagem limitada in vivo 22, 23, 24, 25. No modelo de câmara de janela, uma câmara com uma janela transparente é implantado cirurgicamente na pele em difelocais de rendas (pele dorsal ou na orelha, almofada de gordura mamaria, no fígado, etc) no animal (por exemplo, rato, rato, coelho). Enquanto esta abordagem é claramente facilitador de alta resolução em imagiologia in vivo, requer uma cirurgia invasiva para implante da câmara e pode não ser capaz de acomodar os estudos de imagem longitudinais ao longo de semanas ou meses 22. Recentemente, foi demonstrado que a combinação de alta resolução de microscopia confocal com um procedimento minimamente invasivo, isto é, o transplante para a câmara anterior do olho (ACE) fornece uma &quot;janela de corpo natural&quot;, como um. Poderosa e versátil in vivo plataforma de imagem 26, 27 O transplante para o ACE tem sido utilizado nas últimas décadas para estudar aspectos biológicos de uma variedade de tecidos 28, 29, 30, e sua combinação recente com alta resolução de imagem permitiu o estudo da fisiologia das ilhotas pancreáticas, com uma única célula de resolução não- invasiva e longitudinalmente &lt;sup&gt; 26, 27. Esta abordagem foi utilizada para estudar as respostas auto-imunes, durante o desenvolvimento de diabetes do tipo 1 em modelos animais (dados não publicados). Também foi usado para estudar o desenvolvimento do pâncreas, bem como, em estudos de função renal através do transplante de pâncreas em papilas ECA ou individuais nos glomérulos renais, respectivamente (dados não publicados). Um relatório recente usando essa abordagem demonstrou ainda a sua aplicação para estudar as respostas imunes após transplante de ilhotas de pâncreas 31. Importante, este estudo mostrou que o transplante para a câmara anterior do olho fornece uma janela natural do corpo para realizar: (1) longitudinal, de imagem não invasiva de tecidos transplantados in vivo, (2) in vivo cytolabeling para avaliar o fenótipo celular e da viabilidade em situ, (3) acompanhamento em tempo real do infiltrado de células imunitárias no tecido alvo, e (4) a intervenção local por aplicação tópica ou por injecção intraocular. Aqui, nós demonstrate como realizar o transplante para a câmara anterior do olho utilizando ilhotas pancreáticas.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Descrição / Comentários </td></tr><tr><td> IsoTHESIA (isoflurano) </td><td> Buttler Abastecimento Saúde Animal </td><td> 11695-6775-2 </td><td> 99,9% de isoflurano / ml </td></tr><tr><td> Ketaset (ketamina HCL) </td><td> Fort Dodge Animal Health </td><td> 0856-2013-01 </td><td> Anestesia injetável alternativa </td></tr><tr><td> Beprenex (buprenorfina HCL) </td><td> Reckitt Benckiser Saúde (UK) Ltd. </td><td> 12496-075-7-1 </td><td> 0,3 mg / ml </td></tr><tr><td> Eritromicina pomada oftálmica USP, 0,5% </td><td> Akron </td><td> 17478-070-35 </td><td> Aplicada profilaticamente a olho transplantado </td></tr><tr><td> Cloreto de Sódio a 0,9% (Solução salina) </td><td> Hospira Inc. </td><td> 0409-7983-03 </td><td> Para injecção iv. Estéril </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td> 1X. Estéril </td></tr><tr><td> CMRL médio 1066 </td><td> Cellgro </td><td> 98-304 CV- </td><td> Complementada, modificação CIT. Meios preferidos para ilhotas </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

Intravital imagem surgiu como uma ferramenta indispensável na pesquisa biológica. No processo, muitas técnicas de imagiologia têm sido desenvolvidos para estudar diferentes processos biológicos em animais não-invasiva. No entanto, uma das principais limitações na técnica existentes modalidades de imagens intravital é a incapacidade de combinar não-invasivos de imagem, com as capacidades longitudinal de uma única célula de resolução. Mostramos aqui como o transplante para a câmara anterior do olho contorna limitação significativa tal que oferece uma plataforma versátil experimental que permite a não-invasivos de imagem, com a resolução longitudinal celular in vivo. Demonstramos o procedimento de transplante em ratinhos e proporcionam resultados representativos utilizando um modelo com relevância clínica, nomeadamente o transplante de ilhéus pancreáticos. Além de permitir a visualização directa de uma variedade de tecidos transplantados para a câmara anterior do olho, esta abordagem fornece uma plataforma para Screedrogas n realizando acompanhamento a longo prazo eo acompanhamento em tecidos-alvo. Devido à sua versatilidade o transplante, o tecido / célula para dentro da câmara anterior do olho, não só as terapias de transplante benefícios, estende-se a outras aplicações in vivo para o estudo de processos fisiológicos e patológicos tais como o cancro e a transdução do sinal ou o desenvolvimento da doença auto-imune.

Existem alguns parâmetros que definem um transplante &quot;bom&quot;. Um transplante é um bom que prossegue sem hemorragia ao fazer a incisão, como pode ser visto no vídeo. Sangramento é impedido / minimizada por penetrar apenas a ponta do bisturi (agulha) para dentro do ACE (figura 3a). Isso também irá ajudar a evitar o contato e punção da íris. Ele também irá garantir uma pequena incisão que vai curar muito bem sem causar nebulosidade da córnea ao longo do tempo (Figura 3c, d).</...

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

Uma nova abordagem combinando transplantação intra-ocular e microscopia confocal permite longitudinal, não-invasivo de imagem em tempo real, com uma única célula de resolução dentro dos tecidos enxertados<em&gt; In vivo</em&gt;. Demonstramos como transplantar ilhéus pancreáticos para dentro da câmara anterior do olho do rato.

Transplante para a câmara anterior do olho para longitudinal, não-invasivo<em&gt; Em vivo</em&gt; Imaging com uma única célula de resolução em tempo real

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

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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 visualizações.  University of Miami Miller School of Medicine. Uma nova abordagem combinando transplantação intra-ocular e microscopia confocal permite longitudinal, não-invasivo de imagem em tempo real, com uma única célula de resolução dentro dos tecidos enxertados In vivo. Demonstramos como transplantar ilhéus pancreáticos para dentro da câmara anterior do olho do rato.

Vídeo: 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.

De alta freqüência Ecocardiografia de alta resolução: primeira evidência de não-invasivos Medida repetida de Strain miocárdico, contratilidade, e regurgitação mitral no coração Isquemia-reperfundidos murino

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

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.

Imagens em tempo real multiespectrais de fluorescência para detecção intra-operatória do linfonodo sentinela em Oncologia Ginecológica

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.

Imagem não invasivo de rejeição do enxerto aguda após Rat Transplante Renal Usando<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.

Desenvolvimento de um ambiente de jogo de áudio baseada Virtual para ajudar com habilidades de navegação em Cegos

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.

Induzida pela quimioterapia Vascular Toxicidade - em tempo real<em&gt; In vivo</em&gt; Imagem de Impairment Vessel

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.

Avaliação não invasiva da eficácia de novos Therapeutics para Intestinal Patologias Usando imagem endoscópica série da Vivo Ratos

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 termogênico pré-adipócitos para transplante em adiposo Depots Tissue

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.

Estendida Time-lapse Imagem intravital do Real-time multicelular Dynamics no microambiente tumoral

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; Imagens do Cerebrovasculature: Relevância para doenças do SNC

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-Arterialized transplante de fígado parcial de rato com na vivo orientado a embarcação 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. ...