Diese Arbeit wurde von AHA SDG 10SDG2630086 Zhao X, RO1-AR061385 Ma J und GO Stipendium RC2AR05896 um Brotto M. unterstützt

<ol>
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Messung der Kontraktionskraft und Ermüdbarkeit ist für die umfassende Beurteilung der Skelettmuskulatur Funktion wichtig. Der Hauptzweck dieses Tests ist es, Veränderungen in Muskelkraft und ermüdend Eigenschaften unter bestimmten pathologischen Bedingungen, wie Sarkopenie und Muskelermüdung zu identifizieren und die Wirkung von Medikamenten / Reagenzien auf Muskelkontraktilität testen. Da die Muskelkraft ist eng mit der intrazellulären Ca 2 +-Freisetzung aus, extrazelluläre Ca 2 +-Eintrag ...

Skelettmuskeln Knochen des Skeletts befestigt und erzeugen kontraktilen Kräfte unter der Kontrolle des zentralen Nervensystems. Elektromechanische Kopplung (ECC) bezieht sich auf den Prozess der Umwandlung eines elektrischen Stimulus an eine mechanische Reaktion. Ca 2 +-Signal ist eine wesentliche Komponente des kontraktilen Funktion in der Skelettmuskulatur. Effektive Ca 2 + Mobilisierung von sarkoplasmatischen Retikulum (SR) ist ein wichtiger Bestandteil für die ECC in den Muskelzellen 1, 2, und Veränderungen in der intrazellulären Ca 2 +-Signalisierung unterliegen den entsprechenden kontraktile Dysfunktion in einer Reihe von Muskelerkrankungen 3-5. Sachgemäßen Beurteilung Muskelkontraktilität ist wichtig und komplementär zu Ca 2 +-Imaging und andere Assays, um Einblicke in Skelettmuskeln Funktion zu erhalten, nicht nur bei der kontraktilen Ebene, sondern auch auf dem kinetischen Niveau. Kraft und Geschwindigkeit kann auch erhalten, um die wichtige Eigenschaft informierenMuskelkraft und der Status der ECC-Prozess unter verschiedenen physiologischen und pathophysiologischen Bedingungen. Diese fruchtbare Gebiet der Forschung hat eine sehr reiche Geschichte und viele Theorien der Muskelkontraktion erschien über zwei Jahrtausende 6. Modernes Muscle Research beginnt wahrscheinlich in 1674-1682 mit der mikroskopischen Beobachtung der Querstreifung und Myofibrillen in Muskelfasern durch Leeuwenhoek 6. Fast ein Jahrhundert später, bemerkte Luigi Galvani that frog Muskel kontrahiert kräftig, wenn ihre Nerven mit Skalpell während einer Funkenentladung von einem entfernten elektrischen Maschine 7-9 berührt. Kontraktion könnte auch durch die Verbindung des Beines Nerv zum Muskel durch einen metallischen Leiter hergestellt werden. Die Einzelheiten des komplexen elektrischen Signalisierung Mechanismus, durch Galvani befürwortet wurden schließlich von Hodgkin, Huxley und Katz in ihrem berühmten Gleichung 10, 11, die das Fundament der Elektrophysiologie wurde formuliert. Die bemerkenswerte Beobachtungen von Ringer über die Auswirkungen von extrazellulärem Ca 2 + auf die Kontraktilität der Frosch Herz-und Skelettmuskulatur 12-15 stellen den ersten großen Schritt in der Anerkennung der Ca 2 + als zentraler Regulator der Muskelkontraktilität 16, 17. Von den 1980er Jahren bis in die Gegenwart ein Platzen der Entdeckungen in der Muskelkontraktilität Feld wurde durch die Einführung der Muskelkontraktilität und Ermüdbarkeit Protokolle in murine Skelettmuskulatur 18 realisiert. Jones und Edwards waren die ersten, dass niederfrequente intermittierende Müdigkeit (exercise-induzierte Reduktion in Kraft) schlagen 19 mit Veränderungen in der ECC Maschinen und nicht die kontraktilen Apparates verbunden war. In den späten 1980er und frühen 1990er Jahren wurden Kolkeck et al 20, Kolbeck und Nosek 21 und Reid 22 mit Zwerchfell von Tiermodellen, die Auswirkungen der Theophyllinen, cortiosterone und freie Radikale auf die Skelettmuskulatur Kontraktilität studieren, während Brooks und Faulkner waren die ersten, die auf Messungen der wiederholten Kraft und Leistungsmessungen in schnell und langsam Muskeln von Mäusen 22 hinweisen. Darüber hinaus waren Lannegren, Westerblad, Lamb und Westerblad die erste direkte Verbindung ex vivo Kontraktilität mit intrazellulären Ca 2 +-Regulation und begann Infragestellung der Rolle der Azidose bei Muskelermüdung 23, 24. Unsere Labors haben sich seit Beginn des Jahres 2000 zum Verständnis neuartiger Gene mit modulatorische und regulatorischen Aufgaben auf Muskel-ECC mit kritischen Rollen in Muskelkontraktilität, Ermüdbarkeit und Alterung durch eine Kombination von intakten Maus Muskelkontraktilität Studien beigetragen, intrazelluläre Ca 2 +-Überwachung in intakt und gehäutet Muskelfasern und molekular-genetische Manipulationen 3-5, 25-29. Hier haben wir die experimentelle Protokoll für die Messung Kontraktilität murine isolierten soleus und extensor digitorum longus (ED detailliertL) Muskeln, die zu entsprechen einer meist langsam oxidative (Typ I und IIa Muskelfasern) und ein meist schnell glyocolytic Muskeln (Typ IIb und IIx Muskelfasern) mit unterschiedlichen kontraktilen Eigenschaften. In diesem Protokoll wurden intakte Muskel-Sehnen-Komplexe isoliert und badete in einem ADI PowerLab Radnotti Kammer-System mit reinem Sauerstoff oder einem Gemisch aus Sauerstoff (95%) und CO 2 (5%) geliefert. Kontraktilen Kräften wurden von elektrischen Stimulationen von einem Grass-Stimulator erzeugt und detektiert mit einem Kraftaufnehmer, der mit einem ADI PowerLab/400 integriert wurde, so dass die Anpassung von Makro-Routinen für die Erfassung, Sammlung, Digitalisierung und Speicherung von Daten zu steuern. Dieser Aufbau kann zu messen Muskelkraft, Muskelkraft, sowie die Kraft-Frequenz-Beziehung, Muskelermüdung, Erholung von Muskelermüdung, Geschwindigkeit und insgesamt kinetischen Eigenschaften der Muskelkontraktion. Darüber hinaus können die Auswirkungen von Medikamenten auf Muskelkontraktion durch diesen Versuchen beobachtet werden. </p&gt; Die Vorteile dieser Methode liegen bei der Beseitigung der neuronalen und vaskulären Komponenten vom Skelettmuskel, die eine direkte Beurteilung der inhärenten Eigenschaften der kontrahierenden Muskel. Darüber hinaus ermöglichen ex vivo Assays Kontraktilität Manipulation der extrazelluläre Milieu umgebenden isolierten Muskeln, die die Verwendung von pharmakologischen Manipulationen verschiedener Ionenpermeation Kanäle und Transporter um ihre physiologischen Rollen für Skelettmuskel Funktion definieren kann. Diese ex vivo-System hat uns erlaubt, vor kurzem entdecken eine deutliche Alternan Verhalten in bestimmten mutante Muskel-Präparaten, die zu veränderten intrazellulären Ca 2 verbunden waren + Handling-Eigenschaften 4. Alternans als schwankende Burst Episoden kontraktile Kraft während der Rückgang Phase der Ermüdung definiert. Während dieser Veranstaltungen kontraktilen Kräfte kurz über seinen früheren Kraftniveau d zu erhöhenährend ermüdend Stimulation, vielleicht, weil entweder mehr Ca 2 + freigesetzt wird oder die kontraktilen Maschinerie geworden empfindlich auf Ca 2 + 30. Behandlung von Cyclopiazonsäure (CPA), einem reversiblen Blocker des sarkoplasmatischen-endoplasmatischen Retikulum Calcium ATPase (SERCA), Koffein, ein Agonist des Ryanodin Kanal (RyR) und wiederholte ermüdend Stimulationen können alle mechanischen induzieren Alternans 4, was darauf hindeutet, dass die Alternans direkt an Verwandte Modulation des EG Kopplung Prozess. Demonstration der Methode zu induzieren und aufzeichnen Mechaniker alternans in in vitro Kontraktilität Setup dient als Beispiel für die vielfältigen experimentellen Parameter, die mit diesem System oder ähnliche erhalten werden, könnten auf der Grundlage individueller Forschungsinteressen zu zeigen. Diese Methode kann von Interesse für die Forscher studieren Muskelphysiologie sein. Ähnliche Setup kann auch für isolierte Skelett muscle-tendon/ligament Komplexen von anderen verwendet werdenanatomischen Stellen, sowie für einzelne Fasern und Muskelstreifen.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> 2-APB </td><td> Tocris </td><td> 1224 </td><td> Blocker aus einer Anzahl von Ca 2 +-Kanäle einschließlich Eintrag SOC und TRP usw. </td></tr><tr><td> SKF96365 </td><td> Sigma </td><td> SKF-96365 </td><td> Blocker aus einer Anzahl von Ca 2 +-Kanäle einschließlich Eintrag SOC und Rezeptor-vermittelten Ca 2 +-Einstrom usw. </td></tr><tr><td> BTP-2 </td><td> Millipore </td><td> 203.890-5MG </td><td> Relativ spezifisch SOC Blocker </td></tr><tr><td> CPA </td><td> Sigma </td><td> C1530 </td><td> Reversible SERCA Blocker </td></tr><tr><td> Koffein </td><td> Sigma </td><td> C0750 </td><td> Schnelles Handeln RyR Agonist </td></tr><tr><td> Radnoti Vier Einheit Tissue Organ Bath-System </td><td> Radnoti </td><td> 159920 </td><td></td></tr><tr><td> Kombination Tissue Support / Stimulationselektrode </td><td> Radnoti </td><td> 160151 </td><td> Vertical Zig Zag Typ mit Gewebe Unterstützung </td></tr><tr><td> Quad-Brücke Amp </td><td> ADInstruments </td><td> FE224 </td><td></td></tr><tr><td> PowerLab/400 </td><td> ADInstruments </td><td></td><td> Dieses Produkt ist nicht mehr verfügbar. Wählen Sie andere Version des Datenerfassungssystem. </td></tr><tr><td> Kraftaufnehmer (5 mg - 25 g) </td><td> ADInstruments </td><td> MLT0201/RAD </td><td></td></tr><tr><td> Diagramm v4.02 </td><td> ADInstruments </td&gt; <td></td><td> LabChart 7,3 ist die neueste Version von Chart-Software. </td></tr><tr><td> S8800 Dual-Pulse Digitale Stimulator </td><td> GRASS TECHNOLOGIES </td><td></td><td> Dieses Produkt ist nicht mehr verfügbar. S88X Dual Output Square Pulse Stimulator ist eine neuere Stimulator. </td></tr><tr><td> RF Transformer Isolation Unit </td><td> GRASS TECHNOLOGIES </td><td> Modell SIU5 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

ex vivo assessment of contractility, fatigability and alternans in isolated skeletal muscles

Hier beschrieben ist ein Verfahren, um die Kontraktilität von isolierten Skelettmuskeln messen. Parameter wie Muskelkraft, Muskelkraft, kontraktile Kinetik, Ermüdbarkeit und Erholung nach Ermüdung erhalten, um spezifische Aspekte der Erregung und Kontraktion Kupplung (ECC) Verfahren wie Erregbarkeit, kontraktilen Maschinen und Ca 2 + Handhabbarkeit zu beurteilen. Diese Methode entfernt die Nerven und Blutversorgung und konzentriert sich auf die isolierten Skelettmuskel sich. Wir routinemäßig verwenden diese Methode, um genetische Komponenten, die die kontraktilen Eigentum der Skelettmuskulatur zu verändern, obwohl modulierenden Ca 2 + Signalwege zu identifizieren. Hier beschreiben wir ein neu identifiziertes Skelettmuskel Phänotyp, dh mechanische Alternans, als Beispiel für die verschiedenen und vielfältigen Informationen, die unter Verwendung der in vitro-Assay kann Muskelkontraktilität werden. Kombination dieses Assays mit einzelnen Zell-Assays, genetische Ansätze und biochemistry Assays können wichtige Einblicke in die Mechanismen der ECC in der Skelettmuskulatur bieten.

Watch this Scientific Journal Video about Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles at JoVE.com

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Wir beschreiben ein Verfahren zur direkten Messung der Muskelkraft, Muskelkraft, kontraktile Kinetik und Ermüdbarkeit der isolierten Skelettmuskulatur in einem<em&gt; In vitro</em&gt; System mit Feld Stimulation. Wertvolle Informationen über Ca<sup&gt; 2 +</sup&gt; Handhabungseigenschaften und kontraktilen Maschinen des Muskels kann unter Verwendung verschiedener Protokolle stimulierenden werden.

Ex Vivo Beurteilung der Kontraktilität, Müdigkeit und Alternans in isolierten Skelettmuskulatur

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

ex-vivo-assessment-contractility-fatigability-alternans-isolated

Research

JoVE Journal

Biology

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

23.7K Aufrufe.  UMDNJ-Robert Wood Johnson Medical School. Wir beschreiben ein Verfahren zur direkten Messung der Muskelkraft, Muskelkraft, kontraktile Kinetik und Ermüdbarkeit der isolierten Skelettmuskulatur in einem In vitro System mit Feld Stimulation. Wertvolle Informationen über Ca 2 + Handhabungseigenschaften und kontraktilen Maschinen des Muskels kann unter Verwendung verschiedener Protokolle stimulierenden werden.

Serie: Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

In-vivo-Mikro-Zirkulation Measurement im Skelettmuskel von Intra-vital-Mikroskopie

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Forschung

Biologie

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

DNA Transfektion von Säuger Skelettmuskulatur mit In Vivo Elektroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

Die Ex vivo Isoliert Skeletal Mikrogefäßdichte Vorbereitung für Untersuchung der vaskulären Reaktivität

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Isometrische und exzentrische Krafterzeugung Beurteilung der Skelettmuskulatur Isoliert von Mausmodellen von Muskeldystrophien

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Bewertung von Calcium in Sparks Intact Skelettmuskelfasern

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Kontraktilität Messungen auf isolierte Papillarmuskeln für die Untersuchung von Herz Inotropie bei Mäusen

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Die c-fos-Protein Immunohistologische Nachweis: Ein nützliches Werkzeug als Marker der Mittelwegen beteiligt sind in spezifischen physiologischen Antworten<em&gt; In Vivo</em&gt; und<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Messung der Steifigkeit von<em&gt; Ex Vivo</em&gt; Maus Aorten Mit Rasterkraftmikroskopie

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

Die Maus isoliert perfundierten Nieren-Technik

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Messung der insulin- und kontraktionsstimulierten Glukoseaufnahme in isolierten und inkubierten reifen Skelettmuskeln von Mäusen

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Ex Vivo Beurteilung der Kontraktilität, Müdigkeit und Alternans in isolierten Skelettmuskulatur

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