Os autores agradecem Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, e Stanford Núcleo de Imagem de Pequenos Animais para assistência técnica. Os autores também agradecem AM Bickford, Inc. para suporte de equipamentos veterinários. Esta pesquisa foi suportada por concessões de pesquisa do National Institutes of Health (R01 HL-75774, R01 CA098303, R21 HL085743, 1K12 HL087746), o Programa de Pesquisa do Tabaco Califórnia Related Disease da Universidade da Califórnia (15IT-0257 e 1514RT-0169) , e do Instituto de Medicina Regenerativa da Califórnia (RS1-00183). NH é apoiado por uma bolsa da American Heart Association.

1. Indução de isquemia dos membros posteriores unilaterais <ol><li> Os instrumentos cirúrgicos necessários para esta operação são: multa fórceps pontiagudo, uma pinça pontiaguda, tesoura primavera, tesouras cirúrgicas, porta-agulhas e afastador. Nós fazemos nossa própria retrator usando um clipe de papel porque ele é menor do que retratores disponíveis comercialmente. Esterilizar essas ferramentas antes da cirurgia por uma autoclave ou um esterilizador hot-pérola. Uma ferramenta de cautério e estéril fino cotonetes apontou também será necessária para esta cirurgia. Recomenda-se que as ferramentas de ser re-esterilizados nas pontas, conforme necessário durante o procedimento. </li><li> Quando as ferramentas estão prontas, coloque o mouse na câmara de indução anestésica contendo 1-3% de isoflurano em oxigênio a 100% a taxa de fluxo de 1l/min. </li><li> Deixe o mouse na câmara de indução até que ele não responde a estímulos externos. Em seguida, retire o animal da câmara de indução. Recomenda-se lavar o anestésico da caixa antes de abrir a tampa, para diminuir a exposição do operador para o isoflurano. </li><li> Em seguida, coloque o animal em decúbito dorsal sobre a mesa pré-operacionais e conectá-lo a um fluxo contínuo de isoflurano. Usando um barbeador elétrico, remover os pêlos do membro posterior. Aplique o creme de depilação para remover completamente o cabelo. </li><li> Posicione o mouse na posição supina sobre uma almofada coberta aquecida na mesa de operação, e conectá-lo a um fluxo contínuo de isoflurano. Ampliar e garantir o membro posterior com um pedaço de fita. Uma vez que o membro posterior é seguro, limpe a pele com três esfrega alternando betadine e álcool. Para o restante do procedimento cirúrgico, o uso de um microscópio de dissecação em 10X ou 20X de ampliação para obter uma visão ampliada da região hindlimb. </li><li> Utilizando uma pinça fina e tesoura cirúrgica, fazer uma incisão na pele, de aproximadamente 1 cm de comprimento, a partir do joelho para a coxa medial. </li><li> Usando tampão fosfato (PBS)-umedecidos multa cotonetes apontou, escove delicadamente afastado tecido adiposo subcutâneo em torno do músculo da coxa. </li><li> Em seguida, aplique o cautério transversalmente ao inciso e dissecar através do tecido de gordura subcutânea para revelar a artéria femoral subjacente. </li><li> Use um afastador para abrir o ferimento e obter uma melhor visualização da vasculatura dos membros inferiores. </li><li> Utilizando uma pinça fina e um cotonete bem apontou, gentilmente furar através da bainha membranosa femoral para expor o feixe neurovascular. </li><li> Em seguida, usando um jogo limpo de uma pinça fina e haste com algodão, dissecar e separar a artéria femoral a partir da veia femoral e do nervo no local proximal, perto da virilha. Após a dissecção, passe um fio de sutura de seda 7-0 debaixo da extremidade proximal da artéria femoral. Oclusão da artéria femoral proximal usando nós dobro. Coloque o empate na embarcação como proximal na ferida quanto possível a fim de deixar tempo para o empate segundo e um segmento de intervenção que será seccionado. </li><li> Separe a artéria femoral da veia femoral no local distal perto do joelho. Passe um fio de sutura 7-0 debaixo da extremidade distal da artéria femoral proximal até a artéria poplítea. Oclusão do vaso usando nós dobro. </li><li> Oclusão da artéria femoral distal com um segundo conjunto de nós dobro apenas proximal ao primeiro conjunto de nós. Este segundo conjunto de suturas serão usados ​​para prender a artéria durante o processo de transação. </li><li> Da mesma forma, para fins de emocionante, oclusão da artéria femoral proximal com um segundo conjunto de nós dobro imediatamente distal ao primeiro conjunto de nós. </li><li> Transecto o segmento da artéria femoral entre os nós distal e proximal com um cotonete fino e pontudo e uma tesoura de mola. Tenha cuidado para não perfurar a parede da veia femoral. </li><li> Remover o retrator e fechar a incisão usando Vicryl 5-0. Estas suturas não precisam ser removidos em um momento posterior, como eles se dissolvem sozinhos. </li><li> Uma vez que a incisão é fechada, colocar o animal em cima de uma almofada coberta aquecida na gaiola de recuperação e monitorar continuamente até que desperto. </li><li> Depois de origem animal se recuperou de uma hora, prossiga com o passo de perfusão do laser Doppler sangue, a fim de confirmar a indução de isquemia. </li></ol> 2. Laser Doppler perfusão sanguínea <ol><li> Para começar o laser Doppler perfusão passo, coloque o mouse na câmara de indução anestésica contendo 1-3% de isoflurano em oxigênio a 100% a taxa de fluxo de 1l/min. </li><li> Deixe o mouse na câmara de indução até que ele não responde a estímulos externos. Em seguida, retire o animal da câmara de indução. Recomenda-se lavar o anestésico da caixa antes de abrir a tampa, para diminuir a exposição do operador para o isoflurano. </li><li> Colocar o animal na mesa pré-operacional conectado ao fluxo contínuo de isoflurano. Em seguida, retire o cabelo da hindlimb usando um barbeador elétrico seguido por creme de depilação, se necessário. </li><li> Após a remoção do cabelo,colocar o animal em uma superfície de 37 ° C aquecidos por 5 minutos sob fluxo contínuo de isoflurano. Monitorar a temperatura do núcleo para garantir euthermia, como mudanças de temperatura irá afetar significativamente a perfusão. </li><li> Após 5 minutos, colocar o animal em decúbito dorsal sobre uma superfície não-reflexiva de absorção de luz, como pano de cor verde, conectado a um fluxo contínuo de isoflurano. Estender membros posteriores. </li><li> Em seguida, ligue a impressora a laser Doppler e do software de aquisição e inicializar o software. Especificar o tamanho do campo de visão e resolução. É melhor para manter o campo de visão e densidade de pixels consistente entre os animais, a fim de tornar a análise futura mais fácil. </li><li> Abra um novo arquivo. Prima start para iniciar a aquisição dos dados de imagem. Geralmente, o gerador de imagens detecta automaticamente a distância do mouse, mas se solicitado, especifique a distância do animal para o gerador de imagens. O laser vai mostrar as fronteiras do campo de visão e, em seguida, começar a adquirir dados. </li><li> Após a aquisição for concluída, a imagem começará a mostrar uma gama de cores que são indicativos do nível de perfusão de sangue para as pernas. As cores podem ser definidas para uma gama específica de perfusão para melhor comparar os dados entre os animais. </li><li> Quando a aquisição de dados estiver concluída, salve o arquivo. </li><li> Em seguida, retornar o animal para a gaiola de recuperação e monitorar o animal continuamente até que desperto. </li><li> Para analisar os dados, use o ajuste de limite no software de análise para subtrair qualquer ruído de fundo. Um limite de 0,2 geralmente é um valor razoável. Selecionar 2 regiões de interesse, ou ROIs, que cobrem cada área hindlimb. A variedade de pontos de referência pode ser usada para padronizar o ROI entre os membros e os animais. Em seguida, determinar a perfusão média e variabilidade no ROI. A diferença de perfusão e perfusão entre o membro isquêmico e membro de controle pode então ser facilmente determinada. </li><li> Este procedimento pode ser repetido para acompanhar as alterações na perfusão hindlimb ao longo do tempo. Em um ponto de tempo desejado, o animal pode ser sacrificado para avaliação da função do tecido e comparação com dados de imagem de perfusão. </li></ol> 3. Resultados representativos: A anatomia da vasculatura hindlimb é mostrado na Figura 1 2. Um diagrama representativo da hindlimb após explante artéria femoral é mostrado na Figura 2. Para confirmar a indução de isquemia para o membro posterior, laser Doppler análise de imagens de perfusão demonstra uma redução dramática no fluxo sangüíneo para o membro isquêmico, em comparação com o membro de controle, como mostrado na Figura 3. <img src="/files/ftp_upload/old/1035_2009_1_26_9/figure1_1035.png" alt="Figura 1" width="600" height="406" /> Figura 1. Anatomy da vasculatura hindlimb. Asteriscos indicam os locais de ligação para a indução de isquemia hindlimb. <img src="/files/ftp_upload/old/1035_2009_1_20_7/Niiyama_HLI_Fig2.png" alt="Imagem 2" width="600" height="450" /> Figura 2. Diagrama Representante mostrando a anatomia do membro posterior após a ligadura da artéria femoral na porção proximal e distal após a remoção da artéria femoral. <img src="/files/ftp_upload/old/1035_2009_1_20_8/Niiyama_HLI_Fig3.png" alt="Figura 3" width="600" height="256" /> Figura 3. Laser Doppler imagens mostrando o fluxo de sangue antes e após a indução de isquemia para o membro posterior esquerda (indicada pela seta).

<ol>
	<li>Huang, N. F., Niiyama, H., De, A., Cooke, J. P., , . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+non-invasive+tracking+of+embryonic+stem+cell-derived+endothelial+cells+for+treatment+of+hindlimb+ischemia.">Transplantation and non-invasive tracking of embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia.</a> J Vis Exp. , (2008).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The anatomy of the laboratory mouse. , (1976).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Dokun, A. O., Keum, S., Hazarika, S., Li, Y., Lamonte, G. M., Wheeler, F., Marchuk, D. A., Annex, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+trait+locus+(LSq-1)+on+mouse+chromosome+7+is+linked+to+the+absence+of+tissue+loss+after+surgical+hindlimb+ischemia.">A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.</a> Circulation. 117, 1207-1215 (2008).</li></ol>

Existem algumas fontes de variabilidade a considerar no planejamento e execução de modelos de isquemia hindlimb. Primeiro, o nível de isquemia pode variar de acordo com a localização da ligadura com relação ao de ramos laterais. Portanto, para a consistência do modelo, os animais devem ser submetidos ao mesmo nível de ligadura arterial. Outra fonte de variabilidade na recuperação isquêmica está relacionada com a idade dos animais, com animais jovens (6-8 semanas) com taxas de recuperação mais rápido e mais completo do que animais mais velhos (8-10 meses de idade), avaliado pela hemodinâmica (ie perfusão do laser Doppler) ou funcionais (teste ergométrico, por exemplo) medidas. Para estudos em que se está avaliando um agente angiogênico, pode ser preferível usar animais mais velhos, porque uma maior diferença entre os grupos pode ser observada com uma intervenção terapêutica. Por outro lado, para estudos em que se está avaliando um fator anti-angiogênico, pode ser preferível utilizar animais mais jovens para maximizar o tamanho do efeito 3. Se realizado corretamente, deve haver o mínimo de complicações tais como hemorragia, infecção ou mortalidade. Se o sangramento ocorre por ruptura acidental da veia femoral ou outros recipientes, com uma pressão moderada de algodão estéril aplicador derrubado ou gaze deve ser aplicado no local da hemorragia até que o sangramento pare. Os animais que apresentem sinais de infecção devem ser tratados com agentes anti-infecciosos. Animais exibindo gangrena significativa pode precisar beeuthanized. Esta complicação é mais comum em animais mais velhos, e em algumas cepas, como camundongos BALB c 4. Além disso, o modelo de isquemia hindlimb pode causar a dor ligeira a moderada. Portanto, os analgésicos, como a buprenorfina ou carprofeno deve ser administrado conforme necessário para o tratamento de dor, de acordo com recomendações do IACUC. Em conclusão, nós demonstramos um método simples e reprodutível para o estudo da PAD em um modelo murino de isquemia hindlimb. O modelo de isquemia hindlimb análise com laser Doppler é um excelente sistema para o estudo de patologias vasculares e de avaliação dos candidatos terapêuticos.

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murine model of hindlimb ischemia 

Nos Estados Unidos, a doença arterial periférica (DAP) afeta cerca de 10 milhões de indivíduos, e é também mundialmente prevalente. Terapias médicas para o alívio sintomático são limitadas. Intervenções cirúrgicas ou endovasculares são úteis para alguns indivíduos, mas a longo prazo os resultados são muitas vezes decepcionantes. Como resultado, há uma necessidade para o desenvolvimento de novas terapias para tratar a PAD. A preparação isquemia murino hindlimb é um modelo de PAD, e é útil para testar novas terapias. Quando comparado com outros modelos de isquemia tecidual como a ligadura da artéria coronária ou cerebral, ligadura da artéria femoral prevê um modelo mais simples de tecido isquêmico. Outras vantagens deste modelo são a facilidade de acesso para a artéria femoral e baixa taxa de mortalidade. Neste vídeo, demonstramos a metodologia para o modelo murino de isquemia hindimb unilateral. Os materiais e procedimentos específicos para a criação e avaliação do modelo será descrita, incluindo a avaliação da perfusão do membro por laser Doppler. Este protocolo também pode ser utilizado para o transplante e não-invasiva de rastreamento de células, o que é demonstrado por Huang et al. 1.

Watch this Scientific Journal Video about Murine Model of Hindlimb Ischemia at JoVE.com

Murine Model of Hindlimb Ischemia

O procedimento cirúrgico para a indução de isquemia hindlimb unilateral é demonstrada, com a confirmação de isquemia por laser Doppler imagens de perfusão.

Modelo murino de isquemia dos membros posteriores

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Research

JoVE Journal

Biology

Murine Model of Hindlimb Ischemia 

57.1K visualizações.  Stanford University. O procedimento cirúrgico para a indução de isquemia hindlimb unilateral é demonstrada, com a confirmação de isquemia por laser Doppler imagens de perfusão.

Vídeo: Murine Model of Hindlimb Ischemia

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

O Modelo de encefalopatia hipóxico isquêmica Perinatal da Isquemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biologia

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Embrionárias com células-tronco derivadas de células endoteliais para tratamento da isquemia dos membros posteriores

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

LAD Ligadura: Um Modelo murino de Infarto do Miocárdio

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

Um Método para o alternante dos membros posteriores NASA modelo tradicional de Descarga em Ratos

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Um modelo de anóxia-inanição por isquemia / reperfusão em<em&gt; C. elegans</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

O murino colina deficiente, Ethionine-completada (CDE) dieta modelo de lesão hepática crônica

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

Um modelo murino de gastrectomia de manga Vertical

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Análise de espectrometria de massa para identificar a ubiquização do CENP-A marcado pelo EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Um modelo murino de uma ferida de queimadura reconstruída com um enxerto de pele aogenética

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

Um modelo cirúrgico modificado de isquemia de membros traseiros em ApoE-/- Ratos usando uma incisão em miniatura

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...