Os autores gostariam de agradecer a Andrew J Dunn, um estudante excelente pesquisa de graduação em nosso laboratório, por sua assistência técnica e cuidar dos animais. O Instituto Nacional de Saúde conceder HD058834 e da Universidade de Missouri Life Sciences Fellowship financiado este trabalho.

Cirurgia pré-Instrumentos / Materiais <ul><li> Cortadores de fio / alicates </li><li> Gaze 2 x 2 &quot;pads </li><li> Almofada de aquecimento </li><li> Prata nitrato de varas </li><li> Luvas cirúrgicas estéreis </li><li> Isoflurano inalatório </li><li> Gluconato de clorexidina 4% (matagal cirúrgica anti-séptico) </li><li> Estéril 2-0 sutura aço cirúrgico </li><li> 25 X 5 / 8 agulha Um medidor (autoclavado) </li><li> Hemostat (autoclavado) </li><li> Sutura aço cirúrgico </li></ul> Procedimento Cirúrgico <ol><li> Criar um campo estéril. Seguir o procedimento estéril. </li><li> Anestesiar mouse com 2-3% de isoflurano. </li><li> Lugar do mouse sobre a almofada de aquecimento. </li><li> Scrub cauda com solução de clorexidina. </li><li> Localize 5, 6 ou 7 º espaço intervertebral (entre duas protuberâncias ósseas na cauda, ​​começam a contar a partir do final de pêlos no corpo, na base da cauda). </li><li> Use hemostat e agulha 25G para fazer um furo piloto entre duas vértebras, para o espaço intervertebral. </li><li> Use o alicate para cortar o fio de aço 10 cm de comprimento. </li><li> Remova a agulha de sutura de aço e inserir através do orifício, 5 cm de sutura em cada extremidade da cauda. </li><li> Cada lado da cauda deve ter + 5 cm de sutura. </li><li> Mergulhe a ponta do polegar até a cauda. </li><li> Use sua mão livre e vento a sutura de aço na ponta de seu polegar e torça as pontas para formar um laço grande. A parte ferida ou circular deve ser + 3 mm de comprimento. </li><li> Retire o seu polegar. </li><li> Pinça um fio de sutura com o hemostat e dobrá-la em um pequeno laço, a fim de que está ligado o mais próximo possível ao torção no loop original de grande porte. </li><li> Cortar fios em excesso, e dobrar o fio torcido para trás. </li><li> Se houver qualquer sangramento em excesso, pode pará-lo com as varas de nitrato de prata. </li><li> Desligue o mouse isoflurano e coloque em uma gaiola única casa para se recuperar de 5-7 dias antes do membro posterior descarga. Os anéis da cauda entalada se mais de um mouse é alojados juntos. </li></ol> 1. Membro posterior procedimento de descarregamento Materiais <ul><li> Tamanho 24/10 X 4 parafusos, um por animal. </li><li> Nylon tamanho porca 10-24, uma por animal. </li><li> Asa castanha tamanho 10 X 24, dois por animal. </li><li> Tamanho da anilha ¼ X 1, duas por animal. </li><li> Giratória de pesca com tamanho Gancho # 6, 1-11/16 &quot;, e um animal por. </li><li> Máquina de costura de metal da bobina. Um animal por. </li><li> Clipe de papel pequeno para S-shaped gancho, um por animal </li><li> Alicates. </li><li> 2 x 2 gaze &quot;. </li><li> 2 &quot;braçadeiras aglutinante de papel preto. </li><li> Fita tape / laboratório duto. </li><li> 1-3/8 &quot;bar em aço galvanizado com fenda que se estende um pouco além do comprimento total da gaiola </li><li> 50cm x 40cm x 20cm gaiola (gaiola de rato). </li><li> Garrafa de água com tubo dobrado. </li><li> Fio de aço inoxidável, em forma de U para apoiar garrafa de água. </li></ul> Setup gaiola <ol><li> Estabelecer as 1-38 &quot;bar em aço galvanizado com fenda por cima da gaiola, e fita-lo temporariamente para baixo com fita adesiva. </li><li> Parafuso 1 porca borboleta para o parafuso de máquina, inserir uma máquina de lavar, e insira o parafuso de máquina através da chapa de aço encaixados ± 15 cm da borda da gaiola. </li><li> Adicionar outra arruela e porca de asa. Aperte as porcas tanto asa, mas não aperte, pois será necessário fazer ajustes posteriores. </li><li> Insira o parafuso de máquina através do orifício centro da bobina. </li><li> Para evitar que a bobina de cair, o parafuso porca de nylon para o final do parafuso da máquina. A bobina deve girar livremente. </li><li> Insira o gancho do giro de pesca através da parte inferior da bobina. </li><li> Use um alicate para formar um gancho em forma de S a partir do clipe. (Corte dois centímetros do clipe de papel para formar um gancho). </li><li> Insira uma ponta do anzol em forma de S através do orifício da peça giratória de pesca e pinch-la fechada com o alicate. </li><li> Coloque o fio em forma de U, em paralelo com o parafuso, no interior do lado da gaiola, e dobre as pontas para trás para suportar uma garrafa de água cheia. </li><li> Inserir a garrafa de água no fio em forma de U, com o spicket virada para baixo, mas não tocando o fundo. </li><li> Dobre os dois &quot;binder clamp aberto, de modo que não pode fechar perto. Apenas a tensão suficiente para manter 3-4 pedaços de comida na mesma sem ele cair. Esta é impedir a força da pinça de esmagamento do alimento, ou reprimir o nariz mouse. </li><li> Coloque 3-4 pedaços de para roedores no grampo ligante. </li></ol> Membro posterior descarga de Mouse <ol><li> Inserir a extremidade aberta do gancho em forma de S através do anel menor do anel da cauda. </li><li> Ajuste a altura do mouse, apertar ou afrouxar a porca inferior da asa no parafuso da máquina. As extremidades inferiores não deve ser rolamento do peso. Uma vez que a altura ideal é alcançada aperte a porca de asa superior. </li><li> Reposicionar a garrafa de água, e em forma de U de fio para que o mouse não pode subir no spicket com seus membros posteriores.<html> Segura a garrafa de água com fita adesiva para o lado da gaiola. </li><li> Posição do grampo fichário com comida no fundo da gaiola para que o mouse não pode subir nela; tape-a com fita adesiva. </li><li> Repita o mesmo procedimento se mais de um animal é descarregado hindlimb do outro lado da gaiola. Casa de dois animais por gaiola, espaço os animais para que eles possam quase se tocam. </li><li> Use a 2 X 2 gaze para enrole frouxamente a cauda do mouse sobre o giro de peixe. Use um pequeno pedaço de fita para envolver a gaze, garantir a gaze para a cauda. Este passo é importante para evitar a cauda de drooping para baixo, levando ao acúmulo de sangue na ponta da cauda e que a cauda se tornar necrótico. </li><li> Adicionar uma pequena quantidade de cama cob para a jaula. Não adicione cama muito caso contrário o mouse de um monte de roupa de cama e tentar carregar hindlimbs na cama. </li></ol> 2. Resultados representativos: Para estabelecer o sucesso da técnica HU cauda anel medimos massa corporal do animal antes da cirurgia anel cauda, ​​e antes e depois HU e recuperação; sóleo massa muscular e massa adrenal em todos os grupos. Peso corporal dos camundongos durante o experimento são mostrados na Figura 1. Peso corporal dos camundongos antes da cirurgia anel da cauda (24,7 ± 3,5 g) não foram significativamente diferentes após 5 dias de recuperação da cirurgia do anel da cauda (24,3 ± 2.9g) antes do HU. Nem o peso corporal significativamente declínio imediatamente após 14 dias de HU (22,7 ± 1,9 g), 28 dias de HU (21,3 + 2.1g) ou depois de 3 dias a partir da recuperação HU (24,0 ± 1,8 g). Embora HU para 14 e 28 dias, resultou em uma pequena perda (-6,8% e 12%), mas não significativo na massa corporal, os ratos totalmente recuperado de sua pré-HU peso corporal após 3 dias de recuperação. Consumo de alimento para ratos e camundongos controle ambulatorial HU é mostrado na Figura 3. Ingestão média diária de alimento não foi diferente em camundongos HU mais de 28 dias em comparação aos controles. Observou-se atrofia muscular significativa sóleo seguintes HU para 14 e 28 dias (-39,1% e -46,6%), indicando um procedimento eficaz HU (Figura 2). Além disso, este grau de atrofia é consistente com os valores esperados para a literatura 2-4 semanas de HU 2, 7, 8. Após três dias de recuperação da HU sóleo massa aumentou significativamente para 74% dos valores controle. Sóleo em massa no grupo de recuperação foi significativamente maior que os grupos de ambos os HU, mas ainda assim significativamente menos do que animais do grupo controle ambulatorial. Adrenal pesos foram medidos como um indicador do estresse animal; ambas as glândulas supra-renais esquerda e direita foram excisados ​​e pesados. Os pesos médios foram comparáveis ​​para o controle de ratos e camundongos HU em todos os grupos (Figura 4). Anteriormente em ratos que mostraram que a taxa de sucesso para o número de animais no início do HU em relação ao número de animais que completou HU sem sair do aparelho de suspensão (devido à mastigação animais através da fita cauda-tração) foi de 10 out de 12 usando a técnica de Morey-Holton 1. Nenhum dos ratos (n = 54) neste estudo já desceu da suspensão antes do término do período HU. Fizemos no entanto, têm um rato que que desenvolveu uma cauda muito inflamada. Quando a inflamação se tornou evidente, nós removemos o anel da cauda para permitir que a área inflamada para curar, e posteriormente colocadas uma outra cauda anel para completar a duração do HU. No passado, quando nós usamos o método Morey-Holton do HU, que normalmente 1-2 eliminar ratos / grupo, porque eles saem do aparelho de suspensão. Com o método de fuga de anel não houve episódios de ratos, inadvertidamente, que sai do aparelho HU durante o período de HU para 14 ou 28 dias (n = 54 ratos). <img src="/files/ftp_upload/2467/2467fig1.jpg" alt="Figura 1" /> Figura 1. Corporal Alterações Peso (média ± DP). Abreviaturas: CON: ratos peso-rolamento controle (n = 76); Pré-HU: todos os camundongos antes de HU (n = 54); HU14: hindlimb ratos descarregado por 14 dias (n = 34); HU28: hindlimb ratos descarregado para 28 dias (n = 20); REC: ratos hindlimb descarregado permitido recuperar gaiola por 3 dias (n = 22). Não houve diferenças significativas no peso corporal foram observadas entre os grupos. <img src="/files/ftp_upload/2467/2467fig2.jpg" alt="Figura 2" /> Figura 2. Atrofia músculo sóleo (média ± DP). Abreviaturas: CON: ratos peso-rolamento controle (n = 76); HU14: hindlimb ratos descarregado por 14 dias (n = 34); HU28: hindlimb ratos descarregado por 28 dias (n = 20) e REC: ratos hindlimb descarregado permitido a gaiola recuperar durante 3 dias (n = 22). * Significativamente diferente (p &lt;0,001) do peso-rolamento de controle (CON). δSignificantly diferentes (p &lt;0,01) do que ambos os grupos HU. 467fig3.jpg &quot;alt =&quot; Figura 3 &quot;/&gt; Figura 3 Consumo Alimentar. (Média ± DP). Ingestão de alimentos médio diário (g / dia) durante peso-rolamento e descarga hindlimb. Abreviaturas: ratos peso-rolamento controle (n = 76); HU: ratos hindlimb unloaded14 - 28 dias. Não houve diferenças significativas entre os grupos CON e HU foram observados. <img src="/files/ftp_upload/2467/2467fig4.jpg" alt="Figura 4" /> Figura 4. Massa Adrenal (média ± DP). Abreviaturas: CON: ratos peso-rolamento controle (n = 76); HU14: hindlimb ratos descarregado por 14 dias (n = 34); HU28: hindlimb ratos descarregado por 28 dias (n = 20) e REC: ratos hindlimb descarregado permitido a gaiola recuperar durante 3 dias (n = 22). Não foram observadas diferenças significativas entre os grupos.

<ol>
	<li>Brown, M., Foley, A. M., Hasser, E. M., Foley, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+hindlimb+unweighting.">An alternative method for hindlimb unweighting.</a> J Gravitational Physiol. 12, 43-50 (2005).</li><li>Fitts, R. H., Metzger, J. M., Riley, D. A., Unsworth, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+disuse:+a+comparison+of+hindlimb+suspension+and+immobilization.">Models of disuse: a comparison of hindlimb suspension and immobilization.</a> J Appl Physiol. 60, 1946-1953 (1986).</li><li>Ingalls, C. P., Warren, G. L., Armstrong, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++transients+in+mouse+soleus+muscle+after+hindlimb+unloading+and+reloading.">Intracellular Ca2+ transients in mouse soleus muscle after hindlimb unloading and reloading.</a> Journal of Applied Physiology. 87, 386-390 (1999).</li><li>Morey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spaceflight+and+bone+turnover:+correlation+with+a+new+rat+model+of+weightlessness.">Spaceflight and bone turnover: correlation with a new rat model of weightlessness.</a> BioScience. 29, 168-172 (1979).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+of+growing+rats:+a+model+for+predicting+skeletal+changes+during+space+flight.">Hindlimb unloading of growing rats: a model for predicting skeletal changes during space flight.</a> Bone. 22, 83-88 (1998).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+rodent+model:+technical+aspects.">Hindlimb unloading rodent model: technical aspects.</a> J Appl Physiol. 92, 1367-1377 (2002).</li><li>Thomason, D. B., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrophy+of+the+soleus+muscle+by+hindlimb+unweighting.">Atrophy of the soleus muscle by hindlimb unweighting.</a> J Appl Physiol. 68, 1-12 (1990).</li><li>Thomason, D. B., Herrick, R. E., Surdyka, D., Baldwin, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+soleus+muscle+myosin+expression+during+hindlimb+suspension+and+recovery.">Time course of soleus muscle myosin expression during hindlimb suspension and recovery.</a> J Appl Physiol. 63, 130-137 (1987).</li></ol>

Não há conflitos de interesse declarados.

Nós avaliamos um método alternativo de ratos hindlimb descarga, o que estamos determinados a ser um grande sucesso. O método de cauda-anel, conforme descrito aqui foi usada a longo prazo de suspensão dos membros posteriores, e estudar HU recuperação a seguir. Em cada uma dessas condições, não havia nenhuma evidência de falta de adaptação como evidenciado pela ingestão de alimentos estável, peso corporal e pesos adrenal de camundongos. Esta cauda anel técnica HU é de fácil execução e pode ser facilmente incorporado com muito pouco prática cirúrgica. Existem várias vantagens da cauda anel método em comparação com o tradicional Morey-Holton 5, 6 método. Primeiro, a pele eo local de implantação do anel em que a cauda sempre são visíveis, por isso, se surgirem problemas, é evidente de imediato, não apenas no final do período de suspensão. Em segundo lugar, os animais não são capazes de sair do aparelho de suspensão a qualquer momento durante HU, eliminando a necessidade de fazer uma determinação de se manter o rato ou não no conjunto de dados. Além disso, os ratos não incomodar o aparelho tocar e quando eles são liberados da HU para a recuperação, eles não interfira com as suas caudas, permitindo uma fase de recuperação contínua seguintes HU. Finalmente, hindlimb ratos descarregado com o método de cauda anel manter seu peso corporal melhor do que os ratos hindlimb descarregado pelo Morey-Holton técnica de tail-tração. Em nossas mãos após 14 dias de HU observou-se uma pequena perda (-6,8%), mas não significativo na massa corporal. Considerando Ingalls et al. Observada uma diminuição significativa -12% do peso corporal em ratos após 14 dias de HU pelo método de Morey-Holton 3. Embora Ingalls não relataram pesos adrenal, parece que o nosso método cauda anel confere menos stress sobre o mouse (pesos adrenal em ratos nosso HU não são diferentes do que os controles) e melhor manutenção do peso corporal ao longo do período hindlimb descarga. Em resumo, um método do anel da cauda de hindlimb descarga foi desenvolvido e testado em camundongos HU para 14 e 28 dias, e em ratos voltaram para a recuperação da gaiola por 3 dias após HU. Com base em pesos adrenal, massa corporal e ingestão de alimentos, os camundongos parecem ter se adaptado bem ao aparelho cauda-anel. A cauda hindlimb anel descarga método é uma alternativa útil, simples para o Morey-Holton hindlimb tradicional técnica de descarga.

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an alternant method to the traditional nasa hindlimb unloading model in mice

O Morey-Holton hindlimb descarga método (HU) é um amplamente aceito National Aeronautics and Space Administration (NASA) baseados em terra modelo para estudar desuso atrofia-nos roedores 4-6. Nosso estudo avaliou um método alternant ao Morey-Holton HU padrão-ouro técnica de tail-tração em camundongos. Cinqüenta e quatro camundongos fêmeas (4-8 mo.) Foram HU por 14 dias (n = 34) ou 28 dias (n = 20). Recuperação do HU foi avaliada após três dias de deambulação gaiola normal após HU (n = 22). Camundongos com idade casadas (n = 76) serviu de suporte de peso controles. Antes de HU um anel da cauda foi formada com um fio de aço 2-0 cirúrgica estéril que foi passado através do 5 º, 6 º ou 7 º espaço inter-vertebral em forma de disco e em um anel a partir do qual os ratos foram suspensos. Localização vertebral para a cauda anel foi selecionado para equilibrar o peso corporal em animais, sem interferir com a defecação. Determinou-se o sucesso desta técnica HU romance de avaliar o peso corporal antes e depois HU, grau de atrofia sóleo, e HU massa adrenal seguinte. Peso corporal dos camundongos antes HU (24,3 ± 2.9g) não alterou significativamente o declínio imediatamente após 14d de HU (22,7 ± 1,9 g), 28d do HU (21,3 + 2.1g) ou após a recuperação de 3 dias (24,0 ± 1,8 g) . Sóleo massa muscular diminuiu significativamente (-39,1% e -46,6%), seguindo HU por 14 dias e 28 dias, respectivamente (p &lt;0,001). Após três dias de recuperação da massa sóleo aumentou significativamente para 74% dos valores controle. Pesos adrenal de ratos HU não foram diferentes em comparação a camundongos controle. O sucesso do nosso método HU romance é evidenciada pela manutenção do peso do animal corpo, comparáveis ​​pesos glândula adrenal, e atrofia sóleo seguintes HU, correspondentes aos valores esperados literatura 2, 7, 8. As principais vantagens deste método HU incluem: 1) facilidade de análise da cauda durante a suspensão, 2) diminuição da probabilidade de caudas cianótica, inflamada, e / ou necrose freqüentemente observados com a cauda de gravação e HU; 3) há possibilidade de camundongos mastigar a tração fita e saindo do aparelho de suspensão; e 4) recuperação rápida e atividade normais imediatamente após a gaiola HU.

Watch this Scientific Journal Video about An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice at JoVE.com

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

Nós desenvolvemos um hindlimb alternant descarga modelo em camundongos. A principal vantagem do nosso método hindlimb descarga cauda anel sobre a convencional Morey-Holton técnica de tail-tração é um procedimento simples e direta que minimiza o estresse sobre o 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 ...

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Research

JoVE Journal

Biology

20.1K visualizações.  University of Missouri, Columbia. Nós desenvolvemos um hindlimb alternant descarga modelo em camundongos. A principal vantagem do nosso método hindlimb descarga cauda anel sobre a convencional Morey-Holton técnica de tail-tração é um procedimento simples e direta que minimiza o estresse sobre o animal.

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

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

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 ...

Biologia

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Um Método Seccionamento melhorado para amostras de tecidos de plantas recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Um método para avaliar os efeitos da bacteriocina na microbiota intestinal de ratos

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Um método simples e eficiente para In Vivo cardíaco-específicas do Gene manipulação por injeção Intramiocárdica em ratos

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

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 ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

Um método de perfusão antegrada para isolamento cardiomiocócito de camundongos

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

A adesão molecular de imagem em rolamento celular por ensaio de pegada de adesão

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Monitoramento Bioluminescente da Sobrevida do Enxerto em Modelo de Transferência Adotiva de Diabetes Autoimune em Camundongos

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Estabelecimento de um Modelo de Fratura Diafisária do Fêmur em Camundongos

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...