Este trabalho foi financiado por subvenções dos Institutos Nacionais de Saúde (NIH) para SSH (R01-DK078640), do Howard Hughes Medical Institute (HHMI), através do Programa de Certificação HHMI / Vanderbilt University em Medicina Molecular para TJW (GRDOT56006779), o Vanderbilt Diabetes Research e Centro de Treinamento (P30-DK020593) eo Digestivo Vanderbilt Disease Research Center (P30-DK058404) fornecendo serviços principais.

1. Prepare Cânula <ol><li> Aqueça uma seção de uma polegada de comprimento da tubulação PE10 com os dedos e estique-lo para que o tubo se torna fina. Nota: o tamanho do vaso ou conduta a ser canulada irá determinar o grau de estiramento necessário. A cânula deve ser bem esticado para ducto biliar lança, mas só podem ser obrigados a ser moderadamente esticada para caber dentro da veia maior portal. </li><li> Cortar o tubo estirado numa diagonal para gerar uma ponta biselada. </li><li> Cortar a outra borda do tubo para criar um segmento de 5-6 cm. </li><li> Inserir um calibre 32, agulha hipodérmica ½ polegada na extremidade não chanfrada do tubo. </li></ol> 2. Preparar seringa com PBS <ol><li> Encher uma seringa de 3 ml com PBS. </li><li> Parafuso da seringa na agulha hipodérmica com anexo puxado PE10 tubos previamente preparado. </li><li> Seringa Flick e empurrar o êmbolo da seringa para garantir que o PBS foi preenchido a agulha e tubagem eque não existem bolhas de ar na seringa. </li><li> Coloque a seringa preparada com PBS em uma seringa porta-moldada de massa de modelar. Isto irá manter o suporte de seringa estável, enquanto o tubo é introduzido na conduta. </li></ol> 3. Outros Set-up <ol><li> Pesar 0,1 g de catalisador para um recipiente pequeno, por exemplo, um poço de uma placa de 24 poços. </li><li> Puxar um ml de resina para uma seringa de 3 ml. Nota: as quantidades relativas de catalisador e resina pode ser ajustada para alterar o tempo de cura. Decrescente quantidade de catalisador pode, no entanto, aumentar o encolhimento lançar e conduzir a um molde de menos desejável. Nota: ao manusear resina, tomar precauções para evitar contato com a resina ou a inalação dos vapores de resina. Use sempre luvas e executar as etapas que envolvem a resina em uma área bem ventilada, como um capuz químico fumos </li><li> Cortar um pedaço de fio de seda de 5,0 a cerca de 5 polegadas a ser usado como uma ligadura. </li></ol> 4. Prepare Rato <ol&gt; <li> Sacrifício do rato adulto. Coloque o mouse em sua parte traseira e abrir a cavidade abdominal. </li><li> Lay mouse sobre dissecar plataforma escopo. Coloque um tubo de 15 ml cônico perpendicularmente debaixo do mouse para aumentar a visibilidade e acessibilidade ao fígado. </li><li> Umedeça um cotonete com PBS e usá-lo para virar o fígado para cima e longe de você para expor o ponto de vista dorsal do fígado, juntamente com a veia porta extra-hepática ou ducto biliar comum. </li><li> Para moldes da veia porta, você pode impedir a coagulação do sangue, através de lavagem da veia com temperatura ambiente PBS. Coloque uma agulha de uma seringa de 3 ml com PBS e inserir a agulha na veia portal extra-hepática, tanto do fígado como você pode. Com cuidado, empurre PBS através na veia portal. Faça um corte na veia comum cardeal para drenar o sangue. Use um cotonete molhado para massagem fígado, melhorando a drenagem de sangue. Este passo não é recomendado para o ducto biliar e pode não ser necessário em todos os sistemas vasculares. Nota: este passo pode também ser performed com paraformaldeído a 4%, em vez de PBS. Utilização de paraformaldeído a 4% pode aumentar a integridade vascular e resultar em uma conversão mais precisa, mais especificamente de menores estruturas vasculares. </li></ol> 5. Canular veia extra-hepático Portal ou ducto biliar comum <ol><li> Fórceps curvos para passar a ligadura de sutura cirúrgica sob a veia porta ou ducto biliar, cerca de ¼ de polegada a partir do fígado. </li><li> Amarre a sutura em um nó frouxo comum. NÃO apertar o nó. </li><li> Se estiver a trabalhar com um sistema aberto, pode ser útil para ligar e apertar uma ligadura na outra extremidade do sistema. Se você abriu a veia comum cardeal para drenar o sangue da veia porta, amarrar uma ligadura ao redor para aumentar a pressão dentro da veia porta e evitar vazamento de resina para a veia hepática central. </li><li> Use uma tesoura de mola para fazer um pequeno corte na veia porta ou ducto biliar, cerca de ¼ de polegada abaixo do nó da sutura. Este cut deve penetrar mais do que metade do diâmetro da veia porta ou ducto biliar. </li><li> Usando fórceps # 5, manter a veia porta ou ducto biliar no local do corte e inserir extremidade chanfrada do tubo no canal biliar. </li><li> Empurrar a ponta do tubo após a ligadura pré-amarrado e para o fígado. </li><li> Teste de precisão canulação injetando PBS através da cânula e assistindo para ver se ele entra na veia porta ou ducto biliar. </li><li> Apertar a ligadura para manter a cânula no lugar. </li></ol> 6. Resina Lançai a veia porta ou vias biliares intra <ol><li> Adicionar pré-medidos ml de resina de 1 a pré-medidos catalisador 0,1 g e misture bem. Evitar a geração de bolhas de ar. </li><li> Desenhar resina-catalisador mistura para dentro da seringa de 3 ml. Remova todas as bolhas de ar delicadamente sacudindo a seringa e expulsando bolhas. Nota: para a fundição de estruturas pequenas, pode ser útil utilizar uma câmara de vácuo para remover as bolhas de ar muito pequenas, a partir de thresina e melhorar a qualidade e elenco. Para permitir o tempo para executar este passo, é necessário para diminuir a proporção de catalisador / resina da sugerida acima, a fim de reduzir a velocidade de cura da resina. </li><li> Recolocá PBS contendo uma seringa de resina contendo uma seringa, deixando a agulha de calibre 32 ligada à cânula. </li><li> Lentamente e empurre resina para a veia porta ou ducto biliar até aumenta a resistência e fígado está cheio. </li><li> Use cotonete molhado para massagear suavemente fígado e incentivar o mesmo preenchimento. </li><li> Remover cânula a partir da estrutura e rapidamente voltar a apertar a ligadura para evitar fugas de resina. </li><li> Permitir rato para colocar o plano à temperatura ambiente durante 20 min, enquanto as curas de resina, em seguida, remover o fígado. </li><li> Continue com o de limpeza ou maceração de fígado. </li></ol> 7. Fígado clara para visualização in situ <ol><li> Fixar o fígado em paraformaldeído a 4% durante a noite a 4 ° C, de balanço. </li><li> Lavar o fígadoem PBS durante pelo menos quatro horas, de balanço. Neste ponto, você pode separar os lobos do fígado, se desejar. </li><li> Desidratar em metanol a 50% e depois em metanol a 100% durante pelo menos quatro horas cada, agitando à temperatura ambiente. </li><li> Limpar o fígado em uma solução 1:2 de álcool benzílico e de benzoato de benzilo (BABB) durante pelo menos 24 horas, de balanço. Vezes mais tempo de lavagem pode ser necessária. Se o fígado não desaparecer completamente após 2-3 dias, voltar a 100% de metanol por 24 horas e repetir BABB clareira. </li><li> Fígado submergir em BABB numa placa de Petri de vidro para imagiologia in situ. </li></ol> 8. Macerar tecido hepático <ol><li> Remover fígado de rato e colocados em um tubo cónico de 50 ml com água. Aguarde 1 hora para a cura da resina completa. Lobos do fígado separado se desejar. </li><li> Deitar fora a água e mover-se fígado de uma garrafa de vidro. Submerge em KOH 15%. Deixar durante a noite à temperatura ambiente. Não balance. </li><li> Com cuidado, despeje fora KOH e lavar elenco em água destilada. Nota: o elenco é very frágil e deve ser tratado com extremo cuidado. Evite interromper o elenco ao adicionar ou remover fluido do tubo. </li><li> Quando elenco resina é limpo, deite cuidadosamente em uma Kimwipe para secar, em seguida, transferir para um recipiente para ser armazenado. </li></ol> 9. Resultados representativos Uma conversão bem sucedida da veia porta ou ducto biliar irá mostrar uma rede contínua de ramos progressivamente menores que se estendem a partir do hilo à periferia do fígado. A Figura 1 mostra uma veia porta moldado como visualizado in situ após BABB compensação. Figura 1A mostra a conversão de todo o lobo hepático esquerdo e a forma e tamanho do molde. A Figura 1B mostra uma vista de perto do mesmo molde, demonstrando a a penetração da resina no interior dos pequenos ramos da veia porta, localizada na periferia do fígado. A Figura 2 mostra um molde da veia porta que tenha sido submetido KOH ma2A ceration. figura mostra o lobo hepático esquerdo inteiro e figura 2B é um close-up dos pequenos ramos periféricos. Essa técnica também tem sido aplicado com sucesso para o ducto biliar intra-hepática e publicado utilizando tanto a compensação BABB e técnicas de maceração KOH 1,2,7. Os problemas comuns incluem incompleta enche, bolhas na resina, e em sistemas de transbordamento ou tecidos circundantes. Figura 3 demonstra a forma de reconhecer um preenchimento incompleto (Figura 3A), bolhas (Figura 3A), e um excesso de resina a partir da veia porta para o veia central (Figura 3B). Todas as imagens são tiradas com uma Leica MZ 16 estereoscópio FA e QImaging RETIGA câmera 4000R. <img alt="Figura 1" src="/files/ftp_upload/4272/4272fig1.jpg" /> Figura 1. Elenco resina BABB-limpo de hepático esquerdo da veia porta lobo. A. fundido Resina mostra uma estrutura hierárquica de ramificação que se estende desde o hilo à periferia do fígado. Elenco resina aparece na cor amarela dentro azulada hepática. B. Um fim acima mostra que de preenchimento de resina estende a pequenos ramos na periferia de fígado. A resina é esbranquiçada através do fígado limpa. Barra de escala = 1 mm. <img alt="Figura 2" src="/files/ftp_upload/4272/4272fig2.jpg" /> Figura 2. Tissue-macerado em resina de hepático esquerdo da veia porta lobo. A. Resina elenco de lobo hepático todo mostra muitos ramos de tamanhos variados. B. Close-up mostra pequenos ramos na periferia do fígado. Empenamento aparecimento de pequenos ramos representa resina de enchimento dos espaços sinusoidais e podem ou não ser visíveis na resina molda e vai aumentar a densidade aparente ramo, se presente. A resina é na cor branca. Barra de escala = 1 mm. Figura 3. Problemas comuns com moldes de resina. A. Uma preenchimento incompleto da veia portal lóbulo é aparente por alguns ou todos os ramos fundido deixando de estender para a periferia do fígado. Este fundido também exibe bolhas, visível como quebras na fundição contínua (seta). B. Devido à proximidade da veia porta e ramos da veia central em alguns lugares, a resina da veia porta, muitas vezes, entrar e encher a veia central. Isto pode ser reconhecido quando existem dois distintos ramos hilares (setas) e duas estruturas ramificadas que possuem diferentes padrões e sobreposições. Discriminação entre erros técnicos e verdadeiras alterações morfológicas pode ser feito de duas maneiras. Em primeiro lugar, a estrutura de molde deveria ser comparada com a estrutura esperada com base na 2-dimensional ou de outros métodos de análise. Segundo, é importante realizar várias repetições, e quando possível, quantificar o structure e realizar a análise estatística para determinar a reprodutibilidade e, se houver uma alteração estatisticamente significativa morfológica entre e dentro experimental diferentes e braços de controlo.

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Não há conflitos de interesse declarados.

Nós descrevemos exemplos específicos de como a veia portal e sistemas de vias biliares intra-hepáticos do fígado pode ser fundido, mas esta técnica pode ser aplicado a virtualmente qualquer outro sistema ductal ou vascular com pequenas adaptações. Os trabalhos anteriores demonstraram a viabilidade desta técnica, em várias espécies, incluindo o mouse 1,2,7-9, patinho 10,11, coelho 12,13, cão 14, e suíno de 15, e em muitos órgãos, inclusive da glândula ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Tubagem de polietileno PE10 </td><td> Fisher Scientific </td><td> 1417012P </td><td></td></tr><tr><td> 5-0 seda preta cirúrgica trançada </td><td> Roboz Surgical </td><td> SUT-15-1 </td><td></td></tr><tr><td> Steriject 32 G x 13 mm agulha </td><td> Ar-tite </td><td> TSK3213 </td><td></td></tr><tr><td> Tesoura Primavera </td><td> Belas Science Tools </td><td> 15000-08 </td><td></td></tr><tr><td> Número 5 fórceps </td><td> Belas Science Tools </td><td> 91150-20 </td><td></td></tr><tr><td> Mercox II kit </td><td> Ladd Research </td><td> 21247 </td><td></td></tr><tr><td> O álcool benzílico </td><td> Fisher Scientific </td><td> 1816-1804 </td><td> Only necessário para BABB-clearing </td></tr><tr><td> Benzoato de benzilo </td><td> MP Biomedicals, LLC </td><td> 154839 </td><td> Só é necessário para BABB-compensação </td></tr><tr><td> Salina tamponada com fosfato (PBS) </td><td></td><td></td><td></td></tr><tr><td> Massa de modelar </td><td></td><td></td><td></td></tr><tr><td> Escala </td><td></td><td></td><td></td></tr><tr><td> Laboratório de tesoura </td><td></td><td></td><td></td></tr><tr><td> 15 ml de polipropileno tubos com tampas de </td><td></td><td></td><td></td></tr><tr><td> Paraformaldahyde 4% </td><td></td><td></td><td></td></tr><tr><td> De hidróxido de potássio 15% (KOH) </td><td></td><td></td><td> Só é necessário para maceração </td></tr><tr><td> Gilete </td><td></td><td></td><td></td></tr><tr><td> Metanol a 100% </td><td></td><td></td><td></td></tr><tr><td> 3 ml de seringa luer lock </td><td></td><td></td><td></td></tr></tbody></table>

3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system

De órgãos, a arquitetura correta de estruturas vasculares e ductal é indispensável para a função fisiológica adequada, e a formação e manutenção de tais estruturas é um processo altamente regulado. A análise destes complexos, três estruturas tridimensionais grandemente dependia tanto exame 2-dimensional da secção ou em estudos de injecção de corante. Estas técnicas, no entanto, não são capazes de fornecer uma representação completa e quantificáveis ​​das estruturas ductais ou vascular que se destinam a elucidar. Em alternativa, a natureza do 3-dimensional moldes de resina plástica permanente gera um instantâneo do sistema e é um novo e técnica amplamente útil para visualizar e quantificar as três estruturas tridimensionais e redes. Uma vantagem decisiva do sistema de fundição de resina é a capacidade de determinar o intacto e ligado, ou comunicar a estrutura, de um vaso sanguíneo ou adesiva. A estrutura do vascular e ducredes mentais são cruciais para o funcionamento do órgão, e esta técnica tem o potencial para ajudar estudo das redes vasculares, e ductal de várias maneiras. Moldagem de resina pode ser usada para analisar a morfologia normal e arquitectura funcional de uma estrutura de luminal, identificar mudanças morfogenéticas do desenvolvimento, e descobrir diferenças morfológicas na arquitectura de tecido entre os estados normais e de doença. Os trabalhos anteriores resina de moldagem utilizado para estudar, por exemplo, defeitos de arquitectura e funcionais do sistema biliar intra-hepático do rato duto que não foram reflectidas em 2-dimensional análise da estrutura 1,2, alterações na vasculatura cerebral de rato um modelo de doença de Alzheimer 3 , portal anormalidades veia no Portal ratos hipertensos e cirróticos 4, etapas de desenvolvimento em maturação linfática rato entre pulmões imaturos e adultos, 5 imediatas alterações microvasculares no fígado de rato, pâncreas e rim em resposta a ferimentos em química6. Aqui apresentamos um método de gerar um elenco resina 3-dimensional de uma rede vascular do mouse ou ductal, com foco específico na veia porta e ducto biliar intra-hepática. Estes moldes podem ser visualizadas através de compensação ou maceração do tecido e pode então ser analisada. Esta técnica pode ser aplicado a praticamente qualquer sistema vascular ou ductal e seriam directamente aplicáveis ​​a qualquer estudo investigar o desenvolvimento, a função, a manutenção, ou lesão de uma estrutura 3-dimensional ductal ou vascular.

Watch this Scientific Journal Video about 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System at JoVE.com

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

Um método de visualização e quantificação da estrutura 3-dimensional do rato da veia porta hepática ou ducto biliar intra-hepático é descrito. Esta técnica de fundição em resina pode também ser aplicada a outros sistemas do tipo ductal ou vascular e permite<em&gt; In situ</em&gt; Visualização ou quantificação de arquitectura intacto um sistema de comunicação.

3-Dimensional Fundição de resina e Imagem da Veia Rato Portal ou Sistema intra-hepática Duto Biliar

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

3-dimensional-resin-casting-imaging-mouse-portal-vein-or-intrahepatic

Research

JoVE Journal

Medicine

17.1K visualizações.  Vanderbilt University. Um método de visualização e quantificação da estrutura 3-dimensional do rato da veia porta hepática ou ducto biliar intra-hepático é descrito. Esta técnica de fundição em resina pode também ser aplicada a outros sistemas do tipo ductal ou vascular e permite In situ Visualização ou quantificação de arquitectura intacto um sistema de comunicação.

Vídeo: 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelagem e imagem 3-Dimensional Invasion cela coletiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Medicina

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Ligadura do ducto biliar em ratos: Indução de Inflammatory Lesão hepática e fibrose por colestase obstrutiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Utilizando os ácidos biliares e multi-fluorados<em&gt; In Vivo</em&gt; Magnetic Resonance Imaging medir Bile transporte do ácido

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

Overaccumulation induzida por Ileectomy biliares no intestino de rato

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Modelo do rato da partição fígado associando e ligadura da veia porta para procedimento encenado Hepatectomy (ALPPS)

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Imagem de perfusão doppler laser no hindlimb mouse

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Um modelo de rato para pancreatite crônica através da infusão TNBS do ducto biliar

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Minimizando o sangramento da veia do portal pós-infusão durante o transplante de ilhotas intrahápticas em camundongos

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

DUCT: Fundição de Resina Dupla seguida de Tomografia Microcomputada para Análise hepática 3D

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

Dissecção do ducto biliar extra-hepático e da vesícula biliar em neonatos de camundongos com nove dias de idade

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...