The work was supported in part by funding from Alberta Livestock and Meat Agency and Natural Science and Research Council Discovery grant to S. D. Proctor.

Todos os procedimentos em animais experimentais descritas, tanto no vídeo e manuscrito foram aprovados pelo Comitê Cuidado e Uso Institucional Animal e seguiu as orientações estabelecidas pelo Conselho Canadense de Animal Care. 1. anestesia cirúrgica e preparo cirúrgico do Pigs Neonatais <ol><li> Em uma antecâmara separada, premedicate 25 kg porcos perto da base do pescoço, com um cocktail de drogas sedativo-anestésico intramuscular contendo: azaperona (0,3 mg / kg), cloridrato de cetamina (10 mg / kg), a dexmedetomidina (15 ug / kg). Nota: Adicionar buprenorfina (0,005-0,02 mg / kg) no coquetel de drogas pré-anestésica para maior controle da dor intra-operatória. </li><li> Anestesiar os porcos com gás para inalação de isoflurano inalado (4-5% de isoflurano em 500 ml-1000 ml / min O 2) usando uma máscara facial. Visualizar as cordas vocais usando um espaço da laringe veterinário (lâmina reta 17-25 cm de comprimento), e aplicar tópica 10% spay lidocaína ao vocalcabos. Permitir que o spray de lidocaína a contactar as cordas vocais 30-60 seg antes da intubação para reduzir a possibilidade de espasmo das cordas vocais e obstrução das vias aéreas. </li><li> Entubar os porcos por meio de um tubo endotraqueal (5,0-7,0 mm de diâmetro interno (ID)) entre as cordas vocais e manter a anestesia com isoflurano gás (0,5-2,0% de isoflurano em 1.000-2.000 ml / min de O2) usando um fechada circuito de reinalação sistema anestésico durante toda a cirurgia. Avaliar o nível de anestesia pelo tom mandíbula, e ambas as respostas reflexas dos pedais e da pálpebra. gás anestésico expirado é eliminado e ventilado fora do centro cirúrgico. </li><li> Limpar a superfície exterior da orelha com uma solução de limpeza cirúrgica 2,0% de clorexidina seguido por uma lavagem com álcool isopropílico a 70%. Com um cateter intravenoso 20 L, catheterize uma veia da orelha para fornecer fluidos intravenosos (solução de lactato de Ringer; 5-10 ml / kg / hr) durante a cirurgia. Garantir um oxímetro de pulso à superfície da mucosa da língua, com medfita iCal para monitorar a frequência cardíaca ea saturação de oxigenação do sangue periférico (SpO2). </li><li> Coloque o porco anestesiado em uma posição reclinada dorsal e raspar o abdômen ventral a partir de meados do tórax caudalmente ao aspecto ventral do púbis. Limpar esta área com os dois alternando 2,0% clorexidina cirúrgico esfrega e lavagens de água estéril. </li><li> Transferir o porco anestesiados ao centro cirúrgico e aplicar a paramentação final de 70% lavagem de álcool isopropílico, deixe-a secar, e depois armar o animal. </li><li> Insira uma sonda de temperatura rectal cerca de 2-4 cm no recto para monitorizar a temperatura do corpo. Coloque os suínos de uma almofada de aquecimento de recirculação de água para manter a temperatura corporal normal (38-40 ° C) durante o procedimento cirúrgico. </li><li> Armar o porco com quatro toalhas cortinas colocadas em um padrão quadrante sobreposta em torno do abdômen. Coloque a primeira cortina em todo processo xifóide do esterno, a segunda cortina junto aspecto lateral do abdômen cerca de 5 cm lateral à linha média abdominal. Coloque a terceira cortina entre a crista do íleo da pelve e a quarta cortina, como a segunda cortina (embora no lado oposto), é colocada ao longo da face lateral do abdómen cerca de 5 cm lateralmente à linha média abdominal. </li><li> Coloque uma grande cortina de mesa, com uma fenda de abertura permitindo o acesso ao local da cirurgia, sobre as cortinas toalha subjacentes e cubra o porco e toda mesa cirúrgica. A cortina final é um Steri-cortina descartável colocado sobre a grande cortina mesa. </li></ol> 2. Cirurgia Abdominal e cateterismo do Tronco linfático intestinal <ol><li> Faça uma incisão na pele 20 cm, com uma lâmina de bisturi para expor os músculos abdominais subjacentes. Inciso as camadas musculares abdominais com electrocauterização mono-polar (20 configuração Watts) para expor o peritoneu parietal. Abra um segmento linear 20 cm de peritônio parietal com uma tesoura Metzenbaum para acessar as vísceras abdominais e vasos linfáticos. </li><li&gt; Coloque um retractor na face craniana da incisão cirúrgica para manter a cavidade abdominal aberta para a duração da cirurgia. </li><li> Humedecer todos os tecidos com água morna (37 ° C) de soro fisiológico estéril por todo o procedimento cirúrgico. Levante cuidadosamente um grande segmento do intestino incluindo o de cólon, ceco, íleo e jejuno da cavidade abdominal e exteriorizar-lo para o flanco esquerdo do porco para acessar a parte superior do abdome, fígado e vasos linfáticos. Fixe o intestino exteriorizado na posição com cortinas toalhas adicionais para formar um estilingue para apoiar delicadamente o intestino. </li><li> Localize o vaso linfático, estabelece aproximadamente 4 cm crânio-medial da veia renal direita, 6 cm ventral caudal-medial- dos capatazes cava e sob o aspecto visceral do lobo direito do fígado perto do pâncreas 22,36,37. Identificar o vaso linfático como uma estrutura transparente justaposto ao segmento ventral direito da veia porta 36,37. </li><li> Separe o Vess linfáticael dos fascia em torno suavemente provocando afastado o tecido anexado com aplicadores Q-tip. Uma vez que os aspectos laterais do recipiente são separados a partir do tecido circundante, criar uma abertura &quot;túnel&quot; debaixo do navio com excelentes sem corte com ponta fórceps. </li><li> Passe três 2-0 suturas de seda por baixo do vaso linfático com uma pinça fina. Ligadura da sutura mais caudal primeira para ocluir, dilatar e encher o recipiente com linfa. Propositadamente deixar as extremidades deste fio de sutura relativamente longo (4 cm), para fixar o cateter para o vaso linfático. Colocar duas outras suturas separadas de 1,0 cm um do outro e são de aproximadamente 1,0-1,5 cm cranianos à sutura caudal garantido. Deixar estas duas suturas com um &quot;laço solto ligadura único&quot; para permitir a fixação mais rápido do cateter para dentro do vaso. Nota: O segmento do vaso linfático localizado entre o fio de sutura ligaï¿½o da sutura e mais caudal do meio (de as duas suturas cranianas) é o local para a cateterização. Em relação sumaterial de ture, poliglactina 2-0 sutura pode substituir o fio de seda 2-0, se necessário. </li><li> Corte um pequeno buraco dentro do vaso com íris tesouras e dilatar o vaso com uma pinça sem corte finas. Inserção de aproximadamente 1,0-1,5 cm de tubo de cateter especializado (4,06 Diâmetro exterior (OD) X 2,31 milímetros ID) com uma extremidade chanfrada para dentro do vaso e amarrar as duas suturas cranianas para fixar o cateter no lugar. Use as extremidades longas de sutura da sutura de caudal para fixar o cateter para o recipiente. </li><li> Lave o intestino exteriorizado com grandes quantidades de solução salina quente e devolvê-lo suavemente para a cavidade abdominal garantindo posicionamento anatômico correto do intestino. </li><li> Exteriorizar o cateter no flanco meados esquerda (5-10 cm ventral da fossa paralombar). Adicione uma incisão na pele com um bisturi e passar um trocarte a partir da cavidade abdominal para a superfície da pele para criar uma abertura para a exteriorização do cateter. Use um grande fórceps de Kelly para exteriorizar o cateter da cav abdominaldade através da abertura trocar. </li><li> Fechar o peritônio parietal com um padrão de sutura simples contínua de poliglactina 2-0 sutura com uma agulha rodada (cônico). Feche as camadas musculares abdominais com um padrão de sutura simples interrompido com uma sutura de poliglactina 2-0 em uma agulha rodada. </li><li> Feche a pele em um padrão subcuticular com poliglactina 2-0 sutura em uma agulha de corte. Fixe o cateter exteriorizada na pele com um padrão de sutura em bolsa e uma sutura 2-0 nylon em uma agulha de corte. </li><li> Coloque um revestimento especializado no porco enquanto ainda anestesiados para facilitar sua colocação e reduzir o estresse do porco durante a recuperação. </li></ol> Recuperação 3. pós-cirúrgica e Cobrança A linfa <ol><li> Cerca de 10 minutos antes da interrupção da anestesia inalatória, administrar bupenorphine (0,1 mg / kg) por via intramuscular para fornecer imediato analgesia pós-cirurgia. Continuar bupenorphine (0,1 mg / kg) a cada 12 h durante 24-48 h, para manteranalgesia pós-cirurgia. </li><li> Monitorar os porcos para complicações pós-cirúrgicas a cada 8-12 horas, por um período de 7 dias. </li><li> Recolha a linfa em 500 ml de frascos de lavagem de polipropileno, revestido com ácido etilenodiamina tetraacético (EDTA), e suplementado com antibióticos; penicilina (6000 UI), estreptomicina (6 mg) e anfotericina B (3 mg) a cada 12 h durante um período de 7 dias. </li></ol> 4. Quantificação de Lipoproteína apoB48, triglicerídeos, colesterol e proteína total arrecadado de linfa <ol><li> Centrifugar a amostra de linfa a 1.800 xg durante 5 min a 4 ° C. Recolhe-se o sobrenadante e usá-lo para a quantificação de triglicéridos, colesterol e proteína total. </li><li> Divide-se o sobrenadante em três amostras: uma amostra não diluída, uma amostra diluída 1:20 água destilada e uma amostra final foi diluído 1: 100 com água destilada. </li><li> Utilizar o sobrenadante não diluído para medir os níveis de colesterol, com um kit disponível comercialmente. </li><li> Use a 1:20 e 1:100 amostras diluídas para medir triglicérido e os níveis totais de proteína com um kit comercialmente disponível e o ensaio de proteína total de ácido bicinconínico respectivamente. </li></ol> 5. Quantificação de Lipoproteína apoB48, coletados a partir de linfa 38 <ol><li> Determinar a concentração de lipoproteína de apoB48 com um método imunológico Ocidental adaptado Blotting 38. linfa total de separada, com 3-8% de dodecil sulfato de sódio a electroforese em gel de poliacrilamida Tris-etilo-(SDS-PAGE). </li><li> Transferir as proteínas separadas para uma membrana de fluoreto de polivinilideno (0,45 um) e incube-as com um anticorpo policlonal de cabra para a ApoB (1: 4000), então ligá-lo com um anticorpo secundário anti-cabra. </li><li> Quantificar a lipoproteína apoB48 com quimiluminescência usando uma comparação densitométrica linear com padrão de proteína apoB48 purificado roedor. </li></ol>

The authors have nothing to disclose.

Coleta de linfa intestinal é um excelente método para investigar mecanismos envolvidos na lipídico 8-12 e 13-15 de drogas metabolismo, a metástase do câncer 16,17, o tráfico de celular e função imunológica 18-26, em vários modelos animais experimentais. Com efeito, a capacidade de colher grandes quantidades de um ou outro periférico (aferente) e central (eferente e grandes vasos do tronco) linfa durante um período prolongado tem sido particularmente importante para...

O sistema linfático é uma área pouco estudada da fisiologia. Modelos pré-clínicos de cateterismo linfático ocorrer em diferentes espécies animais e 1-8 são utilizados pelas indústrias farmacêuticas e instituições de investigação para investigar mecanismos envolvidos em lípidos 8-12 e 13-15 metabolismo da droga, metástase de câncer 16 com tratamento experimental 17, 18 e a função imunológica -26. Este estudo explora o uso de cateterismo intestinal tronco linfático em um modelo de porco doméstico para medir componentes do metabolismo de lipoproteínas. metabolismo das lipoproteínas está envolvido na produção e secreção de quilomicrons, bem como as alterações nos lípidos associados e proteína total. Essas considerações são importantes, pois há grandes diferenças no metabolismo lipídico entre modelos de roedores comumente usados ​​e os seres humanos e, como tal, empregando modelos de suínos para coletar linfa intestinal poderia fornecer informação mais comparável para o estudo de lipídios memetabolização em pessoas 27-31. Várias técnicas cirúrgicas são usados ​​para coletar a linfa intestinal em espécies de animais de grande porte: uma abordagem ombro cranial (ou seja, o cateterismo ducto torácico) 5, uma abordagem lateral superior flanco 32-34, e uma linha média ventral ou abordagem paramediana 22,35. Este vídeo descreve em detalhes o procedimento cirúrgico em suínos utilizando uma abordagem cirúrgica ventral para o cateterismo do tronco linfático intestinal. Cuidadosas autorizações técnicas cirúrgicas este método de cateterismo linfática para recolher grandes quantidades de linfa e dos seus componentes ao longo de períodos de tempo prolongados. Esta técnica abre uma miríade de aplicações para muitas disciplinas que examinam várias funções fisiológicas. Aplicações podem incluir, mas não estão limitados a, todo lipoproteína corpo e o metabolismo lipídico, imunovigilância, génese de tumores e metástases, função intestinal, e a development e progressão da doença inflamatória intestinal.

<table border="0" cellpadding="0" cellspacing="0" width="567">
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			<td height="40" style="height:40px;width:211px;">Miller laryngoscope blade</td>
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			<td style="width:71px;">Zoetis</td>
			<td style="width:119px;">6295</td>
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			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">IPVH00010</td>
			<td style="width:167px;">0.45 &#956;m pore size</td>
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			<td height="40" style="height:40px;width:211px;">ApoB antibody&#160;</td>
			<td style="width:71px;">EMD Millipore</td>
			<td style="width:119px;">AB742</td>
			<td style="width:167px;">1:4000 dilution</td>
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		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Donkey anti-goat IgG-HRP</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">Sc-2304</td>
			<td style="width:167px;"></td>
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long-term catheterization of the intestinal lymph trunk and collection of lymph in neonatal pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

<html> cateterismo linfática do tronco linfático intestinal de suínos neonatal permite a coleta de cerca de 1 L / 24 hr da linfa centro durante um período de 7 dias. A linfa recolhida neste experiências continha componentes do metabolismo de lípidos, proteínas ou seja total de linfa, apoB48 lipoproteína, triglicéridos, proteína total, e colesterol. Tabela 1 destaca valores representativos destes componentes lipídicos de amostras linfáticos agrupados de três porcos. Notavelmen...

Watch this Scientific Journal Video about Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs at JoVE.com

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Long-Term Cateterismo do tronco linfa intestinal e Recolha de linfa no Neonatais Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

long-term-catheterization-intestinal-lymph-trunk-collection-lymph

Research

JoVE Journal

Medicine

8.6K visualizações.  University of Alberta. We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Vídeo: Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

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

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Medicina

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

Básico Técnicas Cirúrgicas na Minipig Göttingen: Intubação, cateterismo vesical, cateterização veia femoral e perfusão Transcardial

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Infarto do Miocárdio e Avaliação de Resultados Funcional em porcos

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Coleta e Extração de DNA de saliva para Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

De alta frequência Ecografia de mouse linfonodos cervicais

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

Cirurgia de colocação de cateteres para Cardiovascular Testes exercício de longo prazo em Suínos

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

Técnica laparoscópica para a coleção de série do fígado e mesentérica linfonodos em macacos

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Análise de Citometria de Fluxo de Subconjuntos de Células Imunes dentro do Baço de Murina, Medula Óssea, Linfonodos e Tecido Sinovial em um Modelo de Osteoartrite

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Analisando gravações de eletrocardiografia de longo prazo para detectar arritmias em camundongos

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Robot-assisted Total Mesorectal Excision and Lateral Pelvic Lymph Node Dissection for Locally Advanced Middle-low Rectal Cancer

Since their approval for clinical use, da Vinci surgical robots have shown great advantages in gastrointestinal surgical operations, especially in complex procedures. The high-quality 3-D visual, multijoint arm and natural tremor filtration allow the surgeon to expose and dissect more accurately with minimal invasion. Total mesorectal excision is the standard surgical technique for the treatment of resectable rectal cancer. To reduce the lateral recurrence rate, lateral pelvic lymph node dissection can be performed, as it is a safe and feasible procedure for locally advanced middle-low rectal cancer with a high possibility of metastasis to the lateral lymph nodes. However, the complexity of the anatomic structures and the high postoperative complication rate limit its application. Recently, several surgeons have increasingly used robotic techniques for total mesorectal excision and lateral pelvic lymph node dissection. Compared with open and laparoscopic surgery, the robotic technique has several advantages, such as less blood loss, fewer blood transfusions, minimal trauma, shorter postoperative hospitalization, and quicker recovery. A robotic approach is generally regarded as a reasonable alternative for complicated procedures such as lateral pelvic lymph node dissection, although there are a limited number of high-quality prospective randomized controlled studies reporting direct evidence. Here, we provide the detailed steps of robot-assisted total mesorectal excision and lateral pelvic lymph node dissection performed at the First Affiliated Hospital of Xi&#39;an Jiaotong University.

The robotic technique described herein aims to detail a stepwise approach to robot-assisted total mesorectal excision and lateral pelvic lymph node dissection for locally advanced (T3/T4) rectal cancer located below the peritoneal reflection.

Excisão total mesorectal assistida por robô e dissecção de linfonodo pélvico lateral para câncer retal médio-baixo localmente avançado

Since their approval for clinical use, da Vinci surgical robots have shown great advantages in gastrointestinal surgical operations, especially in complex ...