טכניקה כירורגית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> החולה מושכב על פי תקן התנאים בחדר הניתוח במצב הגב ועל הבטן כולה desinfected. בשלב הבא, המטופל מכוסה בסדינים סטריליים. </li><li style=";text-align:right;direction:rtl"> לאחר laparotomy &quot;הפוכה בצורת האות&quot; חלל הבטן הוא חקר להוציא הצפק carcinomatosis. בהמשך טרס ligamentum ואת falciforme ligamentum מחולקים עם קשירות electrocautery. </li><li style=";text-align:right;direction:rtl"> בעקבות חקירה של חקר חלל הבטן זהיר דו ידנית של הרצועה הכבד מתבצעת להוציא הלימפה extrahepatic גרורות הצומת. Hilar מבנים extrahepatic ייפגעו אם חדירת הגידול המרכזי או hilus הצומת גרורות הלימפה אינן נכללות. </li><li style=";text-align:right;direction:rtl"> חקר הכבד מתחיל עם מישוש ידני של הכבד מלא, לאחר חילק את הרצועה הכבד ימין משולש. זה ואחריו ultrasonogram intraoperative דופלר לחקור את התפשטות הגידול intrahepatic והקשר שלו וסקולרית המבנים העיקריים intrahepatic, במיוחד באמצע וריד הכבד, וכתוצאה מכך לקבוע ולסמן את קו חיתוך רוחב. </li><li style=";text-align:right;direction:rtl"> אם תהליך הריפוי מופיעה אפשרי, בזכות חמי הכבד מגויס לחלוטין על ידי חלוקת ורידים הכבד האחורי בין קליפים או תפרים מ subphrenium ומן retrohepatic הנחות הווריד הנבוב. שלב זה נמשך עד וריד הכבד הימני הוא חינם. לאחר בידוד וריד הכבד ימין, לולאה mersilene הוכנס לשימוש מאוחר יותר הרמה של הכבד במהלך חיתוך רוחב parenchymal. </li><li style=";text-align:right;direction:rtl"> בשלב הבא, כריתת כיס מרה מדרדר מבוצע. צינור פיברוזיס מתחלקת בין קטעי טיטניום. </li><li style=";text-align:right;direction:rtl"> בהמשך לנתיחה parenchymal הוא התחיל. הכבד הוא החזיק בעדינות, הרים סימטרי פתח עם שתי מגבות. חיתוך רוחב parenchymal מבוצע עם מבתר CUSA (Valleylab, Boulder, CO, ארה&quot;ב). כלי קטן חתומות עם מים מושקים דו קוטבית מלקחיים (Erbe, T בינגן, גרמניה), וכלי גדול או צינורות המרה נחלקים בין חיבורי אותיות, קליפים או אפילו התפרים. כריתות מבוצעות עם לחץ ורידי מרכזי נמוך CVP (0 5 מ&quot;מ כספית) כדי לצמצם את אובדן הדם. שלב זה נמשך עד intrahepatic pedicle הנכונה היא חופשית לחלוטין. ואז, לולאה mersilene מוכנס להרים את pedicle ולאפשר מבוא atraumatic של מהדק של כלי הדם (Autosuture ת&quot;א על ​​ידי Tyco Healthcare Group, Norwalk, קונטיקט, ארה&quot;ב). </li><li style=";text-align:right;direction:rtl"> Pedicle הזכות היא transected עם מהדק ולאחר מכן חילק בצורה חדה את הסכין בצד ימין שלה. </li><li style=";text-align:right;direction:rtl"> ואז שאר חיתוך רוחב parenchymal הושלמה כפי שתואר לעיל על ידי השעיית הכבד באמצעות לולאה mersilene הציב בעבר. </li><li style=";text-align:right;direction:rtl"> בהמשך וריד הכבד הימני הוא ניתח עם מהדק כלי הדם ואת הכבד באונה הימנית מוסר. </li><li style=";text-align:right;direction:rtl"> זה ואחריו הגרסה זהיר של פני השטח חתך בדיקה מדויקת כדי למנוע דליפות מרה. בדיקה לבן ניתן לבצע בשלב זה אם נדרש (7). </li><li style=";text-align:right;direction:rtl"> השלב האחרון הוא קיבעון האנטומי של האונה בכבד שריד ידי תפרים קטע ואז הבטן נסגר על ידי תפרים פועל. </li><li style=";text-align:right;direction:rtl"> לאחר הניתוח המטופלים נצפתה על ביחידה לטיפול נמרץ למשך לילה אחד שגרתי. אם במהלך הניתוח היא אירועים עם sonography נורמלי ותפקוד הכבד חולים נלקחים למחלקה להתאוששות נוספת. </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 מבוצעות בעיקר לאחר הכנת hilar מבנים extrahepatic בתוך הרצועה hepatoduodenal, גם גידולים שפירים או גרורות בכבד. 1-5 במרכזים שונים, חיתוך רוחב parenchymal מבוצע תחת חסימה מוחלטת של כלי הדם או סלקטיבית hilar. החסרונות העיקריים של הליכים אלה הם הכנה מורכב של hilus עם סיכון של פגיעה במבנים הנגדי וכן את האפשרות של דימום מהפורטל בצד ענפי וריד זלוף או לקוי של דרכי המרה. זו טכניקה אפשרית של hemihepatectomy כריתה ימין או של מגזרים לרוחב שמאל עם חיתוך רוחב intrahepatic של pedicle עוזב את הרצועה hepatoduodenal נגעה לחלוטין. גישה זו ניתן להשתמש באותם גידולים בכבד, שם lymphadenectomy מיותר oncologically, וכן סימנים שפיר.

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

כריתה כבדית מוצלח דורש ידע אנטומי עמוק טכניקה כירורגית עדינה. Hemihepatectomies מבוצעות בעיקר לאחר הכנת hilar מבנים extrahepatic בתוך הרצועה hepatoduodenal, גם גידולים שפירים או גרורות בכבד.<sup&gt; 1-5</sup&gt;. Lymphadenectomy extrahepatic האזורית הוא תקן אונקולוגי בבית בסרטן כיס המרה hilar, cholangio הסלולר intrahepatic קרצינומה ו קרצינומה hepatocellular, ואילו הלימפה גרורות בכבד hilus בחולים עם גרורות בכבד הם סמוי לעתים רחוקות. החסרונות העיקריים של הליכים אלה הם הכנה מורכב של hilus עם סיכון של פגיעה במבנים הנגדי וכן את האפשרות של דימום מהפורטל בצד ענפי וריד זלוף או לקוי של דרכי המרה. פיתחנו טכניקה של hemihepatectomy זכות או כריתה של חלקים לרוחב שמאל עם חיתוך רוחב intrahepatic של pedicle זה משאיר את הרצועה hepatoduodenal נגעה לחלוטין.<sup&gt; 6</sup&gt; עם זאת, אם להדמיה intraoperative או מישוש של הרצועה הוא חשוד על חדירת הגידול או גרורות הצומת לימפה, hilus צריך להיחקר ו lymphadenectomy שבוצע.

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

וידאו זה מתאר hemihepatectomy ימין עם חיתוך רוחב intrahepatic של hemipedicle הזכות לעזוב את הרצועה hepatoduodenal נגעה לחלוטין.

Hemihepatectomy העכבר על ידי חיתוך רוחב intrahepatic Suprahilar של Hemipedicle העכבר באמצעות מהדק דם

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

right-hemihepatectomy-suprahilar-intrahepatic-transection-right

Nanyang Technological University

Research

JoVE Journal

Biology

14.0K Views.  Tübingen University Hospital. וידאו זה מתאר hemihepatectomy ימין עם חיתוך רוחב intrahepatic של hemipedicle הזכות לעזוב את הרצועה hepatoduodenal נגעה לחלוטין.

Video: 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.

זיהוי של תחמוצת החנקן אניון קיצוני superoxide ידי ספקטרוסקופיה פאראמגנטיים אלקטרונים תהודה בין תאים באמצעות מלכודות ספין

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

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.

ויזואליזציה של כלי דם Ca 2 + איתות מופעלות על ידי ROS נגזר אוטוקריני

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.

אוסף חלב בעכברוש שימוש נימי צינורות והערכה של שומן חלב תוכן על ידי 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 ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

באמצעות מתאם תמונה דיגיטלי לאפיין זנים מקומיים בדגימות רקמת כלי דם

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

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.

מבדק פשוט עבור הערכת גורמי הגדילה של אנדותל כלי דם

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.

בידוד של תאי שריר חלק Murine כלילית דם

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.

מדידה רגיש של Mitophagy מאת Flow Cytometry באמצעות תלויי-pH הכתב פלורסנט 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.

אינדוקציה של כישלון חדרית הנכון על ידי מכווץ עורק ריאתי והערכה של תפקוד המוח הימני בעכברים

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.

הערכה של תגובתיות וסקולרית באמצעות העורקים המבודדים הבודדים באמצעות התמקדות באפנון על ידי פריסקולרית של הרקמות

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.