This work was supported by National Institutes of Health Grants R00 HL112952 (to R. H.), R01 HL108618 (to J.P.G.), P01 HL051971, and P20 GM104357. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

<ol>
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The authors declare that they have no competing financial interests.

Der vorstehende Protokoll beschreibt die Methoden, um gleichzeitig den Fluss des Substrats durch Glucose-Oxidation und die Oxidation von Fettsäuren in der isolierten Arbeitsrattenherzen zu quantifizieren. Die Messungen können dann auf die aufgezeichneten Herzfunktionsparameter überlagert werden , um die Beziehung zwischen den Substraten Stoffwechsel und Herzarbeit unter der Basislinie und Belastungsbedingungen (Veränderung der Arbeitsbelastung, Ischämie-Reperfusion, etc ...) zu bestimmen. Es ist auch mögl...

Klinische Relevanz Im Säugerherz, gibt es eine starke positive Beziehung zwischen dem Fluß von Substraten durch oxidativen Stoffwechselwege, ATP Erzeugungs- und Herzarbeit 1. In den vergangenen zwei Jahrzehnten hat sich die Untersuchung der komplizierten Verbindung zwischen Herz-Stoffwechsel und Funktion führte zu erkennen , dass Veränderungen in der Herzstoffwechsel eine Ursache für die Kontraktions Dysfunktion sind und möglicherweise pathologischen strukturellen Umbau bei der Festlegung der verschiedenen Arten von Herzerkrankungen 2-4. daher wird erwartet , dass unser Verständnis der Mechanismen metabolischen Umbau des belasteten Herzleitungs wird zur Vorbeugung oder Behandlung von Herzinsuffizienz 5-7 zur Identifizierung therapeutischer Targets führen. Die jüngste Veröffentlichung einer wissenschaftlichen Erklärung der American Heart Association zum Thema &quot;Beurteilung der Herz Metabolism&quot;, betont das wachsende Interesse der wissenschaftlichen Gemeinschaft für tsein Forschungsgebiet 8. Doch während die technologischen Fortschritte in der Herzbildgebung jetzt für eine schnelle und genaue Bewertung der kardialen Morphologie und Funktion zu ermöglichen, die in - vivo - Studie von Herz-Stoffwechsel bleibt begrenzt und beschwerlich: Nuclear Magnetic Resonance (NMR) Spektroskopie und Positronen - Emissions - Tomographie (PET) Bildgebung kann werden verwendet , um Herzenergiereichen Phosphatstoffwechsel und Krebs - Zyklus - Aktivität, aber diese Techniken sind geplagt durch hohe Betriebskosten und durch ihre Unfähigkeit zur Bestimmung des Beitrags der verschiedenen Substrate zu oxidativen Stoffwechsel in stationären Bedingungen 9 .To diesem Zeitpunkt der ex vivo verfolgen Herz Vorbereitung Arbeits stellt die einzige und einzigartige Technik zur Verfügung, gleichzeitig zu studieren und in Echtzeit, Kontraktionsfunktion und Fluss von Substraten in oxidativen Stoffwechselwege 7,9. Das folgende Protokoll zielt darauf ab, Richtlinien für die Herstellung und Verwendung von Reagenzien zu schaffen, verwendet die Ratte zu bestimmen,es von Substraten Nutzung in der isolierten Arbeitsrattenherz. Die isolierte Arbeits Nagetier-Herz-Apparat Obwohl die Technik Hälfte ist fast ein Jahrhundert alt, bleibt die isolierte Arbeitsrattenherz Vorbereitung eine Methode der Wahl für Herz-Kreislauf-Forschung. Wie bei der Langendorff-Herz der Zubereitung bietet das Arbeits rodent Herz eine relativ einfache, zuverlässige und kostengünstige Möglichkeit, eine Vielzahl von Herzparameter unabhängig von den verwirrenden Effekte anderer Organe, neurohormonal und andere zirkulierende Faktoren zu messen. Aber im Gegensatz zu dem Langendorff perfundierten Herzen, das Arbeits Herz weiterhin nahezu physiologischen Herzarbeit, eine Voraussetzung für die Bildung von oxidativen metabolischen Flusses auf ein Niveau durchzuführen , die zu in vivo - Bedingungen relevant sind. Dies wird durch die Bereitstellung der Perfusionspuffer mit dem linken Ventrikel (LV) über eine Kanüle mit dem linken Vorhof erreicht, und da der LV füllt und Verträge, diePuffer wird durch die Aorten-Leitung gegen einen entschlossenen Nachbelastung hydrostatischem Druck ausgestoßen. Das Design der Perfusionsapparatur beschrieben ursprünglich von Neely und Kollegen 10 wurde anschließend durch Taegtmeyer verbessert, Säume und Krebs 11, hat aber nur sehr wenig verändert seitdem. Wie in der ursprünglichen Gerät beschrieben, kann Kontraktionsfunktion durch Bestimmung der Herzleistung beurteilt werden, nicht mehr als Messzylinder mit und eine Stoppuhr Aorten zu messen und koronare fließt 10,11. Mehrere Hersteller bieten nun komplette Arbeits Nagetier Herz Perfusionssystemen. Diese kommerziell erhältlichen Vorrichtung kann mit flowprobes, Druckwandler erfaßt werden, ein Druck-Volumen-Katheter und die gesamte Ausrüstung, die für Herzfunktionsdatenerfassung und -analyse. Die Hersteller bieten eine umfangreiche Dokumentation und Schulungen den neuen Benutzer mit ihrer Ausrüstung vertraut zu machen. Einige Übersichten auch Detail-Protokolle auf dem Arbeits Herz instrumentation und über die Verwendung von Kathetern , Herzfunktion in Nagetieren 12-15 zu messen. Aus diesem Grund werden wir nur kurz auf die Set-up des Perfusionsgerät erwähnen und dem Aufnahmegerät. Das vorliegende Protokoll vielmehr soll die bereits vorhandenen Informationen mit einer Beschreibung der Methoden zu ergänzen, die gleichzeitig durchgeführt werden können, um die Raten der Glukose und langkettigen Fettsäureoxidation, die beiden großen Energie liefert Substrate in der normalen Herz messen. Wir beschreiben hier all die Schritte bei der Verwendung von radiomarkiertem Energiesubstrate für die Beurteilung der myokardialen oxidativen Metabolismus, aus der Herstellung von Reagenzien und Puffer zur Gewinnung und Verarbeitung von Proben, zur Datenanalyse. Prinzipien des Verfahrens Kardiomyozyten erzeugen den größten Teil ihrer Energie für die Kontraktion von der oxidative Phosphorylierung von Fettsäuren (hauptsächlich langkettige Fettsäuren) und Kohlenhydrate (glucose und Laktat). Das Herz hat eine begrenzte sehr energisch Reserven und stützt sich auf eine ständige Zufuhr dieser Energie liefert Substrate aus dem Kreislauf. Der Katabolismus von Glucose durch den glykolytischen Weg ergibt Pyruvat, das dann durch die Pyruvat-Dehydrogenase-Komplex von der inneren Mitochondrienmembran decarboxyliert wird. Langkettige Fettsäuren, extrahiert aus Triglyceriden Albumin oder Lipoprotein Zirkulieren werden zuerst in Acyl-CoA-Molekülen in das Cytosol aktiviert und anschließend in der mitochondrialen Matrix durch die Carnitin-Shuttle transportiert, um die beta-Oxidationswegs ein. Die Acetyl-CoA - Molekülen durch den Katabolismus von Glucose und Fettsäuren , die den Krebs - Zyklus Kraftstoff die Reduktionsäquivalente (NADH und FADH 2) , die durch die Elektronentransportkette , verwendet werden zur Erzeugung der Protonen-Antriebskraft über die innere Mitochondrienmembran zu bauen und ATP durch die Aktivität der ATP-Synthase erzeugen. Wasser und Kohlendioxid sind die End-Nebenproduktedie enzymatischen Reaktionen im Inneren des Krebs-Zyklus stattfindet. Die Lieferung von 14 C- und H- 3 radiomarkierte Substrate (wie beispielsweise 14 C-radioaktiv markiertem Glukose und 3 H-radiomarkierten Ölsäure) zum isolierten Arbeits Herz wird folglich für die Produktion von 14 CO 2 und 3 H 2 O führen, wo quantitativ aus dem koronaren Abwasser zurückgewonnen werden. Die Sammlung von 14 CO 2 wird durch Halten der isoliert perfundierten Herzen in eine abgedichtete Kammer und durch sofortiges Gewinnen des koronaren Abflusses durchgeführt , wie es das Herz verlässt. Eine kleine Anionenaustauschsäule verwendet 3 H 2 O aus dem koronaren Ausfluß zu trennen und zu gewinnen. Die Radioaktivität aus den verarbeiteten Proben wird mit einem Flüssigszintillationszähler gemessen und mit Kenntnis der spezifischen Aktivität der radiomarkierten Substraten verwendet, ist es dann möglich, den Fluss von Glukose und Fettsäure in der individuell QuantifizierenOxidationswege 16,17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>BP366</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>P284</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate (MgSO4*7H2O)</td>
			<td>Fisher Scientific</td>
			<td>M63</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher Scientific</td>
			<td>S233</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>AG 1-X8 resin, chloride form, 100-200 dry mesh size, 500 g</td>
			<td>Bio-Rad</td>
			<td>1401441</td>
			<td>This item can be replaced by purchasing directly the hydoxide form&#160; (see reference below), but this will cost almost 8 times more</td>
		</tr>
		<tr>
			<td>AG 1-X8 resin, hydroxide form, 100-200 dry mesh size, 100 g</td>
			<td>Bio-Rad</td>
			<td>1432445</td>
			<td>Purchasing this item allows to bypass the conversion of the anion exchange resin from the chloride form to the hydroxide form (See section 1.2 of protocol)</td>
		</tr>
		<tr>
			<td>Glass Microanalysis Vacuum Filter Holder</td>
			<td>Fisher Scientific</td>
			<td>09-753-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>S318</td>
			<td>Corrosive. Consult the product MSDS for appropriate handling and storage.</td>
		</tr>
		<tr>
			<td>Gas Dispersion Tube with Fritted Cylinder</td>
			<td>Fisher Scientific</td>
			<td>11-138B</td>
			<td></td>
		</tr>
		<tr>
			<td>Probumin Bovine Serum Albumin Fatty Acid Free, Powder</td>
			<td>EMD Millipore</td>
			<td>820027</td>
			<td>We recommend the use of a charcoal-defatted BSA, as other purification process such as cold ethanol fractionation may leave residues toxic for the heart.</td>
		</tr>
		<tr>
			<td>Sodium Oleate</td>
			<td>Sigma-Aldrich</td>
			<td>O7501</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic Acid, [9,10-3H(N)]-</td>
			<td>PerkinElmer</td>
			<td>NET289005MC</td>
			<td>Radioactive material. Follow your Institution&#39;s radiation safety office guidelines for ordering and handling.</td>
		</tr>
		<tr>
			<td>Dialysis Membrane Tubing, 29 mm diameter</td>
			<td>Fisher Scientific</td>
			<td>08-667E</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose, D-[14C(U)]-</td>
			<td>PerkinElmer</td>
			<td>NEC042B005MC</td>
			<td>Radioactive material. Follow your Institution&#39;s radiation safety office guidelines for ordering and handling.</td>
		</tr>
		<tr>
			<td>Humulin R U-100</td>
			<td>Eli Lilly and Company</td>
			<td>NDC 0002-8215-01 (HI-210)</td>
			<td></td>
		</tr>
		<tr>
			<td>Inactin Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>T133</td>
			<td>Controlled substance on USDEA Schedule III</td>
		</tr>
		<tr>
			<td>3-0 Silk Black Braid</td>
			<td>Roboz Surgical</td>
			<td>SUT-15-3</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Hyamine Hydroxide</td>
			<td>PerkinElmer</td>
			<td>6003005</td>
			<td>Highly toxic and causes severe burns. Consult the product MSDS for appropriate handling and storage</td>
		</tr>
		<tr>
			<td>20 mL Glass Scintillation Vials</td>
			<td>Fisher Scientific</td>
			<td>03-341-25E</td>
			<td>Use glass vials for quantitative recovery of 14CO2</td>
		</tr>
		<tr>
			<td>20 mL HDPE Scintillation Vials</td>
			<td>Fisher Scientific</td>
			<td>03-337-23B</td>
			<td>Use HDPE vials for quantitative recovery of 3H2O</td>
		</tr>
		<tr>
			<td>Red Rubber Sleeve Stoppers</td>
			<td>Fisher Scientific</td>
			<td>14-126DD</td>
			<td>Fit 20 mL scintillation vials; Reusable</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 23G x 40 mm</td>
			<td>BD</td>
			<td>305194</td>
			<td>Use to inject perchloric acid through the rubber sleeve stopper of the CO2 trap</td>
		</tr>
		<tr>
			<td>Perchloric Acid, 60%</td>
			<td>Fisher Scientific</td>
			<td>A228</td>
			<td>Highly corrosive and may act as an oxidizer and/or cause an explosion hazard. Consult the product MSDS for appropriate handling and storage</td>
		</tr>
		<tr>
			<td>Ultima Gold, Scintillation Cocktail</td>
			<td>PerkinElmer</td>
			<td>6013327</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Wool</td>
			<td>Fisher Scientific</td>
			<td>AC38606</td>
			<td></td>
		</tr>
		<tr>
			<td>Decon Dri-Clean Detergent Powder</td>
			<td>Fisher Scientific</td>
			<td>04-355</td>
			<td>For cleaning of glassware, plastic parts, and tubing</td>
		</tr>
		<tr>
			<td>Alconox Tergazyme Enzyme-Active Powered Detergent</td>
			<td>Fisher Scientific</td>
			<td>16-000-115</td>
			<td>For cleaning of &#34;hard to reach&#34; surfaces (tubing, glassware) contaminated by fatty acid-BSA residue</td>
		</tr>
	</tbody>
</table>

methods for the determination of rates of glucose and fatty acid oxidation in the isolated working rat heart

Die Säuger Herz ist ein großer Verbraucher von ATP und erfordert eine ständige Energiezufuhr für Substrate Kontraktion. Es überrascht nicht, Veränderungen der myokardialen Stoffwechsel wurden zur Entwicklung der Kontraktions Dysfunktion und Herzinsuffizienz verknüpft. Daher sollte auf einige der Mechanismen beleuchten den Zusammenhang zwischen Stoffwechsel und Kontraktion entwirren Herz Anpassung oder Fehlanpassung bei Krankheitszuständen regeln. Die isolierte Rattenherz arbeitet Zubereitung kann folgen verwendet werden, gleichzeitig und in Echtzeit, Herzkontraktionsfunktion und Energiefluß Substrate in oxidativen Stoffwechselwege bereitstellt. Das vorliegende Protokoll zielt darauf ab, eine detaillierte Beschreibung der Verfahren zur Herstellung und Verwendung von Puffern für die quantitative Messung der Geschwindigkeiten der Oxidations für Glukose und Fettsäuren zur Verfügung zu stellen, der Hauptenergiesubstrate des Herzens bereitstellt. Die Verfahren zur Probenanalyse und Dateninterpretation verwendet werden ebenfalls diskutiert.Kurz gesagt, ist die Technik , die auf die Lieferung von 14 C- radiomarkierten Glucose und einem 3 H radioaktiv markierten Fettsäure langkettige zu einem ex vivo schlagendes Herz über normothermic kristalloider Perfusion. 14 CO 2 und 3 H 2 O, Ende Nebenprodukte die enzymatischen Reaktionen in der Nutzung dieser Energie liefert Substrate beteiligt sind, werden dann quantitativ aus dem koronaren Ausflusses gewonnen. Bei Kenntnis der spezifischen Aktivität der radiomarkierten Substraten verwendet wird, ist es dann möglich, individuell auf den Fluss von Glukose und Fettsäure in der Oxidationswege quantifizieren. Kontraktionsfunktion des isolierten Herz kann mit dem entsprechenden Aufnahmegeräte und direkt korreliert metabolischen Flusswerte parallel bestimmt werden. Die Technik ist sehr nützlich, um den Stoffwechsel / Kontraktionsverhältnis in Antwort auf verschiedene Stressbedingungen, wie beispielsweise Veränderungen in pre und nach der Belastung und Ischämie zu untersuchen, ein Medikament oder ein circulating Faktor oder Produkt nach der Veränderung der Expression eines Gens.

Zwei repräsentativen Experimente sind in den Figuren unten beschrieben ist. In beiden Fällen wurde das Herz eines 16 Wochen alten männlichen Sprague-Dawley-Ratten isoliert und in den Arbeitsmodus mit KH-Puffer nach dem vorhergehenden Vorschrift hergestellt perfundiert. In jedem Experiment wurde das Herz mit einem Spannungszustand unterworfen Herzarbeit zu beeinflussen. Herzkontraktionsfunktion wurde durch kontinuierliche Aufzeichnung von Pulsdruck durch Einsetzen eines Druckwandlers i...

Watch this Scientific Journal Video about Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart at JoVE.com

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

Methoden zur Bestimmung von Preisen von Glucose und die Oxidation von Fettsäuren in der isolierten Arbeitsrattenherz

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

methods-for-determination-rates-glucose-fatty-acid-oxidation-isolated

Research

JoVE Journal

Biology

14.6K Aufrufe.  University of Mississippi Medical Center. The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

Serie: Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Die Implantation von Engineered Tissue in der Ratte Herz

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Forschung

Biologie

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualisierung des schlagenden Herzens in Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an impending predator attack.  Although it is not known whether invertebrates posses an autonomic nervous system identical to that of vertebrates, evidence shows invertebrates have a  sympathetic-like  response to regulate the internal environment and ready the organism to act behaviorally to a given stimuli.  Furthermore, this physiological response can be feasibly measured and it acts as a biological index for the animal's internal state.  Measurements of the physiological response can be directly related to internal and external stressors through changes in the central nervous system controlled coordination of the cardio-vascular and respiratory systems.   More specifically, monitoring heart and ventilation rates provide quantifiable measures of the stress response not always behaviorally observed. Crayfish are good model organisms for heart and ventilatory rate measurements due to the feasibility of recording, as well as the rich history known of the morphology of the crayfish, dating back to Huxley in 1888, and the well-studied typical behaviors.

Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.

Maßnahmen der Herz-und Ventilatorische Preise bei frei laufenden Crayfish

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Auswertung der räumlichen Verteilung der γH2AX folgenden ionisierender Strahlung

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

Quantifizierung der Mikrogefäßdurchlässigkeit im Einzel isolierten Blutdurchbluteten Lungen Rat Vorbereitung

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Bestimmung der Fettsäureoxidation und Lipogenese in Maus primären Hepatozyten

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Nagetier Arbeits Herz Modell für die Untersuchung der myokardialen Leistung und Sauerstoffverbrauch

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Methoden zur Bestimmung von Preisen von Glucose und die Oxidation von Fettsäuren in der isolierten Arbeitsrattenherz

Zusammenfassung

Weitere Videos entdecken

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Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Maßnahmen der Herz-und Ventilatorische Preise bei frei laufenden Crayfish

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

Auswertung der räumlichen Verteilung der γH2AX folgenden ionisierender Strahlung

Quantifizierung der Mikrogefäßdurchlässigkeit im Einzel isolierten Blutdurchbluteten Lungen Rat Vorbereitung

Bestimmung der Fettsäureoxidation und Lipogenese in Maus primären Hepatozyten

Nagetier Arbeits Herz Modell für die Untersuchung der myokardialen Leistung und Sauerstoffverbrauch