Técnica Cirúrgica <ol><li> O paciente é bedded sob condições padrão sala operatória em decúbito dorsal, e todo o abdome é desinfectados. Em seguida, o paciente é coberto com lençóis esterilizados. </li><li> Depois de &quot;reverse em forma de L&quot; laparotomia da cavidade abdominal é explorado para excluir carcinomatose peritoneal. Posteriormente, o ligamento redondo eo ligamento falciforme são divididos com ligaduras e eletrocautério. </li><li> Exploração da seguinte cavidade abdominal exploração bi-manual cuidadosa do ligamento hepática é realizada para excluir extra-hepáticas metástase linfonodal. As estruturas extra-hepáticas hilar permanecem intocados se a infiltração do tumor central ou metástases hilo linfáticos são excluídos. </li><li> Exploração fígado começa com palpação manual do fígado completo, depois de ter dividido o ligamento triangular hepático direito. Isso é seguido por uma ultra-sonografia Doppler intra-operatório para investigar disseminação intra-hepática do tumor e sua relação com as estruturas intra-vascular principal, especialmente a veia hepática média e, conseqüentemente, para determinar e marcar a linha de transecção. </li><li> Se um procedimento de cura parece possível, o direito hemi-fígado está completamente mobilizada a partir da subphrenium e da retro-hepático da veia cava inferior, dividindo as veias do fígado posterior entre os clipes ou suturas. Este passo continua até a veia hepática direita é livre. Após o isolamento da veia hepática direita, um loop Mersilene é inserido para posterior levantamento do fígado durante a transecção do parênquima. </li><li> Em seguida, uma colecistectomia retrógrada é realizada. O ducto cístico é dividido entre os clipes de titânio. </li><li> Posteriormente dissecção do parênquima é iniciado. O fígado é realizado com cuidado, simetricamente levantou e abriu com duas toalhas. A transecção do parênquima é executada com um Dissector CUSA (Valleylab, Boulder-CO, EUA). Pequenos vasos são selados com água irrigada bipolar fórceps (Erbe, T Bingen, Alemanha), e navios de maior porte ou das vias biliares são divididos entre ligaduras, clips ou mesmo suturas. Ressecções são realizados com baixa pressão venosa central CVP (0 5 mm Hg) para reduzir a perda de sangue. Esta fase continua até que o intra-direito do pedículo é totalmente gratuito. Então, um laço Mersilene é inserida para levantar o pedículo e permitir a introdução atraumática de um grampeador vascular (Autosuture TA por Tyco Healthcare Group, Norwalk-Connecticut, EUA). </li><li> O pedículo direito é seccionado com o grampeador e depois dividiu com a faca em seu lado direito. </li><li> Então o resto da transecção do parênquima é concluído como descrito anteriormente, suspendendo o fígado usando o laço Mersilene previamente colocado. </li><li> Posteriormente, a veia hepática direita é dissecada com o grampeador vascular eo lobo direito do fígado é removido. </li><li> Isto é seguido pela revisão cuidadosa da superfície de corte e exploração precisa excluir vazamentos de bile. Um teste em branco pode ser realizada neste momento, se necessário (7). </li><li> O último passo é a fixação anatômica do lóbulo do fígado remanescente por pontos interrompidos e, em seguida o abdome é fechado por sutura contínua. </li><li> No pós-operatório os pacientes são observados na unidade de cuidados intensivos para uma noite de rotina. Se o curso de pós-operatório sem intercorrências, com ultra-sonografia é normal e pacientes da função hepática são levados para a enfermaria para recuperação posterior. </li></ol>

<ol>
	<li>Belghiti, J., Noun, R., Zante, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+triad+clamping+or+hepatic+vascular+exclusion+for+major+liver+resection.+A+controlled+study.">Portal triad clamping or hepatic vascular exclusion for major liver resection. A controlled study.</a> Ann Surg. 224, 155-161 (1996).</li><li>Man, K., Fan, S. T., Ng, I. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+evaluation+of+Pringle+maneuver+in+hepatectomy+for+liver+tumors+by+a+randomized+study.">Prospective evaluation of Pringle maneuver in hepatectomy for liver tumors by a randomized study.</a> Ann Surg. 226, 704-711 (1997).</li><li>Wu, C. C., Hwang, C. R., Liu, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+and+limitations+of+prolonged+intermittent+ischaemia+for+hepatic+resection+of+the+cirrhotic+liver.">Effects and limitations of prolonged intermittent ischaemia for hepatic resection of the cirrhotic liver.</a> Br J Surg. 83, 121-124 (1996).</li><li>Figueras, J., Llado, L., Ruiz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+versus+selective+portal+triad+clamping+for+minor+liver+resections:+a+prospective+randomized+trial.">Complete versus selective portal triad clamping for minor liver resections: a prospective randomized trial.</a> Ann Surg. 241, 582-590 (2005).</li><li>Lesurtel, M., Selzner, M., Petrowsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Should+Transection+of+the+Liver+Be+Performed?+A+Prospective+Randomized+Study+in+100+Consecutive+Patients:+Comparing+Four+Different+Transection+Strategies..">How Should Transection of the Liver Be Performed? A Prospective Randomized Study in 100 Consecutive Patients: Comparing Four Different Transection Strategies..</a> Ann Surg. 242, 814-823 (2005).</li><li>K&#246;nigsrainer, I., Steurer, W., Witte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+resection+without+hilus+preparation+and+with+selective+intrahepatic+hilus+stapling+for+benign+tumors+and+liver+metastasis.">Liver resection without hilus preparation and with selective intrahepatic hilus stapling for benign tumors and liver metastasis.</a> Langenbecks Arch Surg. 392, 485-488 (2007).</li><li>Nadalin, S., Li, J., Lang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+White+test:+a+new+dye+test+for+intraoperative+detection+of+bile+leakage+during+major+liver+resection.">The White test: a new dye test for intraoperative detection of bile leakage during major liver resection.</a> Arch Surg. 143, 402-404 (2008).</li></ol>

Hemihepatectomies são na sua maioria realizados depois de preparar as estruturas extra-hepáticas hilar dentro do ligamento hepatoduodenal, mesmo em tumores benignos ou metástase hepática. 1-5 Em vários centros, a transecção do parênquima é realizada sob oclusão completa ou seletiva hilar vascular. Principais desvantagens destes procedimentos são a preparação complexa do hilo com o risco de ferir estruturas contralateral ea possibilidade de sangramento da veia porta lateral-ramos ou comprometiment...

right hemihepatectomy by suprahilar intrahepatic transection of the right hemipedicle using a vascular stapler

Ressecção hepática de sucesso requer profundo conhecimento anatômico e técnica cirúrgica delicada. Hemihepatectomies são na sua maioria realizados depois de preparar as estruturas extra-hepáticas hilar dentro do ligamento hepatoduodenal, mesmo em tumores benignos ou metástase hepática.<sup&gt; 05/01</sup&gt;. Extra-hepáticas linfadenectomia regional é um padrão oncológico no colangiocarcinoma hilar, carcinoma intra-celular cholangio e carcinoma hepatocelular, enquanto que metástases linfonodais no hilo hepático em pacientes com metástase hepática raramente são ocultas. Principais desvantagens destes procedimentos são a preparação complexa do hilo com o risco de ferir estruturas contralateral ea possibilidade de sangramento da veia porta lateral-ramos ou comprometimento isquêmico de vias biliares. Nós desenvolvemos uma técnica de hemihepatectomy direita ou ressecção dos segmentos lateral esquerdo com transecção intra-hepática do pedículo, que deixa o ligamento hepatoduodenal completamente intactas.<sup&gt; 6</sup&gt; No entanto, se intra-visualização ou a palpação do ligamento é suspeito de infiltração do tumor ou metástase linfonodal, o hilo deve ser explorada e uma linfadenectomia realizada.

Watch this Scientific Journal Video about Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler at JoVE.com

Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

Este vídeo descreve um hemihepatectomy direita com transecção intra-hepática da hemipedicle direito de deixar o ligamento hepatoduodenal completamente intactas.

Hemihepatectomy direito por Transecção intra-hepática Suprahilar do Hemipedicle direito usando um grampeador Vascular

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after ...

right-hemihepatectomy-suprahilar-intrahepatic-transection-right

Research

JoVE Journal

Biology

14.0K visualizações.  Tübingen University Hospital. Este vídeo descreve um hemihepatectomy direita com transecção intra-hepática da hemipedicle direito de deixar o ligamento hepatoduodenal completamente intactas.

Vídeo: Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Detecção de Óxido Nítrico e ânion radical superóxido por espectroscopia de ressonância paramagnética eletrônica a partir de células usando armadilhas para spins

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Biologia

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Visualização dos Vascular Ca 2 + Sinalização Disparado por parácrina ROS Derivado

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Coleta de Leite no Rato Usando Capilar Tubos e Estimativa da gordura do leite por conteúdo crematócrito

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

A Bioensaio simples para a avaliação dos fatores de crescimento endotelial vascular

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Isolamento de Murino vasculares coronários células musculares lisas

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

Medição de Mitophagy sensível pelo repórter fluorescentes Flow Cytometry usando o pH-dependente mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

Indução de insuficiência ventricular direita por constrição da artéria pulmonar e avaliação da função ventricular direita em camundongos

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Avaliação da responsividade do tônus vascular usando artérias mesentéricas isoladas com foco na modulação por tecidos adiposos perivasculares

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Modificação genética de cianobactérias por conjugação usando o kit de ferramentas de clonagem modular CyanoGate

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37&#176;C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R&#39;s. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

Modelo de sangramento da transeção da veia da cauda em hemofilia totalmente anestesiada um camundongo

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...