The authors acknowledge all colleagues who made this work possible; Ann Demarr&#233;, Veerle Laurysens, Jan De Jonge and Erik Quartier for technical assistance. Financial support was from the VUB Research Council, the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), the Beta Cell Biology Consortium (BCBC), the Fund for Scientific Research Flanders (FWO), Diabetes Onderzoek Nederland (DON) and Interuniversity Attraction Pole (IAP).

Todas as manipulações seguir as orientações emitidas pela Convenção Europeia para a Protecção dos Animais Vertebrados Utilizados para Fins Experimentais e outros Fins Científicos (ETS 123 e e 2010/63 / UE). 1. Preparação de Área de Trabalho <ol><li> Fornecer uma área de preparação dedicada, uma área cirúrgica e uma área de recuperação. </li><li> Conduzir todo o procedimento cirúrgico numa câmara de fluxo laminar, para minimizar os contaminantes ambientais. </li><li> Monte os suprimentos (conforme listado em Materiais e Métodos) necessários na área de preparação, cirúrgica e recuperação, utilizando uma técnica asséptica adequada. </li><li> Certifique-se de que as ferramentas cirúrgicas são autoclavados antes da cirurgia. </li><li> Fornecer uma almofada de aquecimento de água em recirculação, a uma temperatura de 38 ° C para estabilização da temperatura durante a cirurgia. Cubra a almofada de aquecimento com uma almofada impermeável estéril. </li><li> Fornecer um microscópio cirúrgico com uma ampliação de pelo menos 6.3x. </li><li> Use um instrumento sterilizer, tal como um esterilizador quente talão, para esterilizar instrumentos entre os procedimentos cirúrgicos. </li><li> Prepare uma área de recuperação consiste em uma gaiola grande, ladeada por cama plana de papel </li></ol> 2. Preparação dos Animais para Cirurgia <ol><li> Para PDL e cirurgia simulada, utilizar 8 semanas de idade, ratinhos machos, mantidos em gaiolas padrão e mantidos num ciclo luz / escuro de 12 horas 12 horas de luz e alimentados com uma dieta ad libitum roedor padrão. Nota: Aqui, usamos ratos BALB / cJrJ mas nós também têm utilizado com sucesso outras estirpes e várias cepas transgênicas. </li><li> Use Buprenophine como analgesia preventiva (0,05-0,1 mg / kg) 30 minutos antes da cirurgia. </li><li> Anestesiar os ratos por injecção intraperitoneal de 100 mg / kg de cetamina e 5 - 10 mg / kg de xilazina. </li><li> Avaliar anestesia adequada, observando a perda gradual de movimento voluntário e relaxamento muscular. Teste a perda de reflexos por beliscar dedo do pé. </li><li> Aplicar pomada oftálmica para prevent secura dos olhos, enquanto sob anestesia. </li></ol> 3. Preparação do Sítio Cirúrgico <ol><li> Desinfetar tórax e abdômen com solução de clorexidina anti-séptico. </li><li> Raspar uma área de 2,5 cm x 1,5 cm do abdômen. </li><li> Desinfetar a área de barbeado utilizando gaze embebida em solução de clorexidina, em seguida, solução de álcool e uma aplicação final da solução de clorexidina. </li><li> Posicionar o animal na área cirúrgica de modo que o local da cirurgia é preparado para cima voltado para o cirurgião. </li><li> Armar o rato utilizando um penso cirúrgico com uma abertura à prova de água que sai da região abdominal desinfectada exposta ao cobrir o restante do corpo para criar um campo estéril de trabalho. Monitorar o rato antes do procedimento para a profundidade de anestesia. </li></ol> 4. pâncreas ligadura de ducto <ol><li> Laparotomia <ol><li> Faça uma incisão na linha média superior na pele que se estende do processo xifóide ao umbigo usando um estérillâmina cirúrgica. Separe a linha alba subjacente e do peritoneu utilizando tesoura estéril, a fim de expor o quadrante abdominal superior. </li><li> Evitar a secagem-out dos órgãos internos por aspersão regular com estéril de cloreto de sódio a 0,9%. </li><li> Usando pinças estéreis ou zaragatoa, retrair o estômago superiormente, expondo o baço e o lóbulo esplénica (região da cauda) do pâncreas. </li><li> Suavemente retrair o duodeno e o jejuno parte superior para o quadrante superior direito abdominal para expor a cabeça, pescoço e na região do corpo do pâncreas abrangidos pelo peritoneu visceral. </li></ol></li><li> Ligadura <ol><li> Localizar a posição anatómica do ducto pancreático principal na região do pescoço do pâncreas. </li><li> Faça uma incisão na peritônio visceral eo ligamento gastrocolic, a concessão de acesso ao retroperitônio, expondo a região do corpo e cauda do pâncreas. A fim de expor a região do pescoço, executar uma manobra de Kocher. Levante o duodeno e cabeça deo pâncreas fora do retroperitônio elevando-os a partir da veia cava inferior e aorta abaixo. </li><li> Com cuidado, coloque a espátula debaixo região do pescoço. Ligadura do ducto pancreático com fio prolene 6-0 no lado esquerdo da veia porta que separa os lobos gastro-duodenais e esplênicas. </li><li> Executar uma segunda ligadura em estreita proximidade com a primeira ligadura para assegurar que os lóbulos são adequadamente separados. </li><li> Ligadura com muito cuidado para não danificar os vasos sanguíneos subjacentes, nomeadamente a artéria pancreaticoduodenal superior, a artéria pancreaticoduodenal inferior ea parte pancreático da artéria esplênica. </li><li> Colocar os órgãos de volta para a cavidade abdominal. </li><li> Fechar a incisão utilizando 4-0 poliglicol fio filamentoso em um padrão de sutura contínua para o músculo / camada peritoneal e com um padrão descontínuo de sutura para a pele. </li></ol></li></ol> 5. Sham Cirurgia <ol><li> Execute todas as etapas de comodescrito nas etapas 1 a 4.1.4. Enquanto o pâncreas está exposto, não realizar a ligadura do ducto pancreático. </li><li> No final do passo 4.1.4, colocar os órgãos internos de volta para a cavidade abdominal. </li><li> Fechar a incisão, tal como descrito em 4.2.7. </li></ol> 6. Cuidados Pós-operacional e Monitoramento <ol><li> Após o procedimento cirúrgico estiver concluída, coloque o mouse na área de recuperação. Este é constituído por uma gaiola colocada sobre uma almofada de aquecimento e forrado com papel de cama plana, a fim de manter a temperatura normal do corpo. </li><li> Fornecer suporte nutricional para evitar a hipoglicemia pós-operatório por alimentos umedecido colocado no fundo da gaiola. Para esta finalidade, utilizam pastilhas roedor dieta padrão embebidas em água até que amaciar. </li><li> Fornecer suporte de fluido com a comida umedecido e fornecer água ad libitum. </li><li> Use Buprenophine como analgesia (0,05-0,1 mg / kg) duas vezes ao dia durante 2 dias pós-cirurgia. Durante todo o experimental, acompanhamento, observe os ratos de ocorrência de possíveis sinais de infecção, incluindo a secreção de líquido ou pus da ferida, ou por deterioração física caracterizada pela redução na preparação comportamento e nível de atividade, menor apetite e perda de peso corporal. </li></ol> 7. Avaliação do PDL bem sucedido e Colheita de PDL cauda e Sham cauda do pâncreas <ol><li> Euthanize ratos por deslocamento cervical. </li><li> Re-raspar a região abdominal para evitar carry-over de pele. </li><li> Abra a camada de pele e músculo abdominal e remover uma grande área de pele e músculo para se obter um bom acesso para o pâncreas. </li><li> Usando pinças estéreis ou mecha, o estômago é retraído superiormente, expondo o baço e o lóbulo esplénica (região da cauda) do pâncreas. </li><li> Retire cuidadosamente o duodeno e parte do jejuno superior para expor o lobo gastro-duodenal (região da cabeça) do pâncreas. Nota: A porção ligada do pâncreas PDL tem agora reduzido em tamanho e se tornou quasetranslúcido para que ilhotas são visíveis como pequenos pontos brancos. A porção de cabeça do pâncreas é rosa opaco e exócrina distinta lobuli podem ser observados. </li><li> Com uma tesoura estéreis, separar a parte da cauda do pâncreas ligado a partir do baço, por corte ao longo do baço. Cortar o tecido conjuntivo solto ligação entre a região da cauda do pâncreas para os órgãos internos. </li><li> Cortar região da cauda do PDL bem na frente da ligação, excluindo a ligadura eo tecido imediatamente adjacente a ela a partir do tecido colhido. </li><li> Para isolar o tecido cauda sham, siga os passos 7.1 a 7.5. Usando uma tesoura estéreis separar região da cauda do pâncreas simulada a partir do baço por corte ao longo do baço. Isolar a região da cauda por corte na região do pescoço do pâncreas e corte solta do tecido conjuntivo que liga o pâncreas cauda para os órgãos internos Nota: Tanto a cauda e região da cabeça do pâncreas sham são rosa opaco, para isolar as duas partes separadamente,cortar o pâncreas na região do pescoço. </li></ol>

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The authors have nothing to disclose.

In the present study, we describe in detail the methodology behind PDL, a mouse injury model to study beta cell neogenesis and proliferation and transdifferentiation of pancreatic non-beta cells. Ambiguity in data on PDL among labs stimulates the need for a standardized protocol for PDL surgery.
Critical steps in the PDL protocol include the selection of healthy, young mice for surgery. Preferably male mice should be used since unpublished data from our lab suggest that estrogen receptor signa...

O aumento da prevalência de diabetes, que afeta mais de 300 milhões de pessoas em todo o mundo 1,2 impulsionou a busca por novas fontes de células beta produtoras de insulina, tanto in vitro como in vivo, para reabastecer a massa de células beta deficiente. 3 Identificando chave mecanismos e factores que regulam a proliferação de células beta e beta neogénese célula, isto é, a diferenciação das células beta a partir de uma célula ou de células progenitoras não-beta, podem proporcionar novos alvos para o desenvolvimento de terapias de regeneração na diabetes. No pâncreas de roedores em desenvolvimento, todos os tipos de células endócrinas diferenciar a partir de uma população transitória de células progenitoras endócrino, que expressa o factor de transcrição Neurogenin3 (Ngn3) 4,5. No pâncreas roedor adulto, sob condições fisiológicas normais, a massa de células beta é mantido a um número ideal para atender às demandas metabólicas. Alterações na dimensão da célula beta,apoptose e replicação constituem os principais mecanismos para a expansão de células beta e turn-over. 6-8 Embora o potencial de células beta para proliferar sob condições fisiológicas normais não é homogénea em toda a população, 9 a sua taxa de proliferação é baixo e re-replicação é restrito por um período dinâmico quiescência ou período refractário 6,7, influenciada pela idade e o metabolismo da glicose. 10 Uma vez que as células progenitoras endócrina não têm até agora sido identificados no pâncreas adulto normal, neogénese Pensa-se que não contribuem para o crescimento das células beta adulto normal. 8 Portanto, a identificação de uma célula progenitora endócrino facultativa no pâncreas adulto que é expansível e capaz de produzir novas células beta iria proporcionar um novo, possivelmente fonte ilimitada de células beta. Parcial ligadura adesiva (PDL) é um modelo de lesão animal que tem sido descrito para induzir neogenesi célula betas no pâncreas adulto 11,12 Neste modelo., o ducto pancreático principal de drenagem da cauda do pâncreas é ligado cirurgicamente. A obstrução resultante da drenagem exócrina induz maior remodelação de tecidos, acompanhados de inflamação e atrofia acinar distal à ligadura 12-14. Dentro deste ambiente inflamatório, re-expressão do marcador endócrino progenitoras Ngn3 é induzido e o volume da célula beta aumenta duplo . Esta duplicação em beta resultados de volume de células a partir da geração de novas células beta de um Ngn3 expressar-tipo embrionário de células endócrino progenitor e da proliferação de células beta pré-existentes e recém-formados que são propensas a re-duplicação sem &quot;período de quietude&quot;. 11,15 Neogénese células beta e replicação em modelos de lesão, como pancreatectomy 6,7,16-19 e ablação seletiva das células beta 20 têm sido amplamente descrito. No entanto, o resultado regenerativo em these modelos é influenciada pela extensão do dano infligido e está associada com uma massa celular beta inicial diminuiu 21. PDL representa um modelo cirúrgico em que a massa de células beta inicial não é afectado e a neogénese e proliferação das células beta são activados de forma robusta. Com efeito, no pâncreas de ratinhos que foram submetidos a PDL, células que expressam Ngn3 são identificados perto do forro epitelial do ducto. Estas células podem ser isoladas a partir do pâncreas de ratinhos transgénicos ligados Ngn3-GFP usando fluorescência de células activadas (FACS) e são capazes de se diferenciar em relação a células beta funcionais seguinte enxerto em cultura ex vivo e do pâncreas de E12.5 Ngn3 - / - . Ratinhos com 11 Do mesmo modo, em Ngn3 CreERT;. 15 ratinhos R26 YFP em que as células activadas que o gene Ngn3 são permanentemente marcadas após injecção tamoxifeno, Ngn3 células derivadas de células beta do rótulo-positivo são detectados após PDL Além disso, as células beta recém-formados diluir pré -existing células beta e localizar preferencialmente em pequenas ilhotas dentro de células beta que apresentam um elevado potencial de proliferação. Ngn3 15 é importante para a expansão das células beta após PDL desde diminuição da expressão Ngn3 utilizando ARN-gancho de cabelo curto específico para o alvo diminui significativamente a massa de células beta e proliferação das células beta depois PDL. 11 Em particular, a fracção de células derivadas de células beta Ngn3 e a massa celular beta após PDL depende criticamente do nível de indução Ngn3 15. Isto está de acordo com a observação de que o alto nível de expressão Ngn3 é um passo crítico para o compromisso endócrino do pâncreas progenitores multipotentes durante o desenvolvimento do pâncreas 22 Além disso, de ablação seletiva das células que expressam Ngn3 por administração difteria, toxina para Ngn3 CreERT;. Ratos R26 IDTR 15 resulta em diminuição do teor de insulina e reduziu a proliferação das células beta, especialmente em pequenas ilhotas. <pclass = &quot;jove_content&quot;&gt; Embora a indução da expressão Ngn3 em células do duto após PDL foi confirmada por muitos 11,15,16,23,24, expressão Ngn3 em ilhotas células 24,25 e discrepâncias nos resultados de PDL desafiou nossas observações iniciais do aumento da massa de células beta 26,27, aparecimento de Ngn3 expressar progenitores derivados do ducto da glândula endócrina 24,26,28,29 e aumento da proliferação de células beta após 27 PDL 30. Estes resultados conflitantes poderia, pelo menos parcialmente, ser atribuída a uma combinação de factores, incluindo as variações nos pontos de análise, o peso corporal, sexo e idade dos ratinhos, condições fisiológicas e ambientais pós-operatórias, e tempo de pós-cirúrgicos, mais importante, diferenças na técnica cirúrgica. 30 Em nossas mãos, a proliferação de células beta, o conteúdo de insulina, o volume da célula beta e o número de pequenas ilhotas são consistentemente aumentou após PDL. TambémNgn3 mRNA aumenta constantemente, mas há grandes diferenças na expressão de mRNA Ngn3 entre pancreases cauda PDL, para os quais temos nenhuma explicação direta. Colocámos a hipótese de que o nível de ARNm Ngn3 pode correlacionar-se com o grau de neogénese das células beta a partir de células não-beta 15, mas este necessita de mais comprovação. Apesar de não remover todas as variações experimentais, um método padronizado para realização de cirurgia de PDL permite uma melhor uniformidade de resultados e abre novos caminhos para estudar a proliferação de células beta e neoformação.

<table border="0" cellpadding="0" cellspacing="0">
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			<td>Supplies for preparation area&#160;</td>
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			<td>18000-45</td>
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			<td>35968</td>
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			<td>Mini Plasco 0,9% NaCl solution</td>
			<td>B.BRAUN</td>
			<td>3521680</td>
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			<td>6-0 prolene suture</td>
			<td>Ethicon</td>
			<td>8706H</td>
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			<td>4-0 polyglycol suture</td>
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			<td>Taqman Ngn3 primer</td>
			<td>Integrated DNA technologies</td>
			<td>Mm.PT.56a.33574796.gs</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman CycloA primer</td>
			<td>Integrated DNA technologies</td>
			<td>Mm.PT.39a.2.gs</td>
			<td></td>
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		<tr>
			<td>anti-Rabbit Cleaved Caspase 3 antibody (D175)</td>
			<td>Cell Signaling</td>
			<td>5A1E</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse/rat Ki67 antibody</td>
			<td>eBioscience</td>
			<td>14-5698-82</td>
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		<tr>
			<td>monoclonal anti-actin alpha-Smooth muscle-Cy3 (mouse)</td>
			<td>Sigma</td>
			<td>085K4889</td>
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			<td>anti-rat Cytokeratin 19&#160;</td>
			<td>DSHB</td>
			<td></td>
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		</tr>
		<tr>
			<td>monoclonal Mouse anti-BrdU</td>
			<td>Dako</td>
			<td>M0744</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Sigma</td>
			<td>33342</td>
			<td></td>
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surgical injury to the mouse pancreas through ligation of the pancreatic duct as a model for endocrine and exocrine reprogramming and proliferation

Expansão de células beta pancreáticas in vivo ou ex vivo, ou a geração de células beta por diferenciação de uma célula estaminal embrionária ou adulto, pode proporcionar novas fontes de células expansíveis beta para aliviar a escassez doador em transplante de ilhéus humanos como terapia para a diabetes. Apesar de recentes avanços têm sido feitos no sentido de este objectivo, os mecanismos que regulam a expansão das células beta e diferenciação de uma célula-tronco / progenitoras continuam a caracterizar-se. Aqui, nós descrevemos um protocolo para um modelo de lesão no rato adulto pâncreas que pode funcionar como uma ferramenta para estudar os mecanismos de remodelação tecidual e proliferação de células beta e diferenciação. Parcial ligadura adesiva (PDL) é uma lesão induzida experimentalmente do pâncreas roedor envolvendo ligadura cirúrgica do ducto pancreático principal, resultando em obstrução da drenagem de produtos exócrinas para fora da região da cauda do pâncreas. O dano infligido induz atrofia acinar, infilt células imunitáriasração e remodelação do tecido grave. Descrevemos previamente a activação de neurogenin (NGN) 3 expressando células progenitoras endógenas semelhantes e um aumento na proliferação das células beta após PDL. Portanto, PDL fornece uma base para estudar sinais envolvidos na dinâmica das células beta e as propriedades de um progenitor pâncreas endócrino em adulto. Desde então, ele ainda permanece em grande parte não está claro, quais fatores e vias de contribuir para a neoformação de células beta e proliferação no PDL, um protocolo padronizado para PDL irá permitir a comparação entre laboratórios.

PDL induz atrofia acinar e inflamação, mas não afeta o peso corporal ea glicemia Em oito semanas de idade do sexo masculino ratinhos BALB / c, a conduta de drenagem das enzimas exócrinas da cauda do pâncreas é ligado, enquanto a cabeça do órgão, localizada adjacente ao estômago e duodeno, permanece inalterada. Idade, sexo e do sexo masculino camundongos BALB / c da mesma peso submeter à cirurgia sham recapitulando todas as etapas da cirurgia ligadura dos ductos pa...

Watch this Scientific Journal Video about Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation at JoVE.co

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Lesão cirúrgica para o mouse Pâncreas através da ligadura da pâncreas Duct como um modelo para endócrina e exócrina Reprogramação e Proliferação

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

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Research

JoVE Journal

Medicine

16.0K visualizações.  Vrije Universiteit Brussel. This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation. 

Vídeo: Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Um modelo murino de isquemia-reperfusão, através da ligadura da artéria Descendente Anterior Esquerda

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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 ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Infusão do ducto pancreático: um método eficaz e seletivo de medicamentos e entrega viral

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissecação do pâncreas Mouse para análise histológica e perfis metabólicos

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Pancreaticojejuntomia lateral robótica para pancreatite crônica

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Pancreatectomia esquerda radical laparoscópica para câncer pancreático: estratégia cirúrgica e vídeo técnico

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Modelo de Lesão de Isquemia/Reperfusão de Pulmão de Rato em Transplante de Pulmão de Rato: Otimizações para Melhorar os Resultados

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Visualização de nível celular longitudinal estabilizada do pâncreas em um modelo murino com uma janela de imagem intravital pancreática

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

Estabelecimento de um modelo de pancreatite aguda grave do rato usando injeção retrógrada de taurocholate de sódio no ducto biliopancreático

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Ligadura parcial do nervo ciático: um modelo murino de dor neuropática crônica para estudar o efeito antinociceptivo de novas terapias

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...