Questo lavoro è stato sostenuto da AHA SDG 10SDG2630086 di Zhao X, RO1-AR061385 a Ma J e GO di Grant RC2AR05896 di M. Brotto

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Misurazione della forza contrattile e affaticabilità è importante per la valutazione complessiva della funzione muscolare scheletrico. Lo scopo principale di questo test è quello di identificare i cambiamenti nella forza muscolare e le proprietà affaticanti in determinate condizioni patologiche, come sarcopenia e l&#39;affaticamento muscolare, e per testare l&#39;effetto dei farmaci / reagenti sulla contrattilità muscolare. Dal momento che la forza muscolare è strettamente correlato con intracellulare Ca 2 + libero, extracellulare Ca 2 + ingresso e il crosstalk tra i due, possiamo anche raccogliere informazioni su Ca 2 + segnalazione stato nel muscolo scheletrico utilizzando questo metodo. Qui abbiamo mostrato un fenotipo unico chiamato &quot;alternanza meccanico&quot; quando episodi scoppio fluttuanti di forza contrattile durante la fase di declino del profilo faticosa sono stati visti nel quadro di un SR sovraccarico e instabile. Ci si potrebbe aspettare che una varietà di fenotipi muscolo scheletrico compresa la forza alterato, FF, affaticabilità, cinetica contrattili e recupero ability dopo fatica può essere rilevato utilizzando il saggio in vitro contrattilità in diverse condizioni patologiche. La fase più critica per questo saggio è quello di isolare intere muscoli intatti (o fasci muscolari e strisce muscolari) privi di danni. Tale obiettivo è più facile da realizzare in preparazioni muscolo intero. Qui mostriamo che lasciare abbastanza legamenti e dei tendini di legare i muscoli tutto è molto importante per evitare legare il muscolo stesso, che porta ad un danno muscolare e infine la morte del muscolo. Inoltre, bolle costante di soluzioni sia con O 2 al 100% nel caso di HEPES soluzioni a base di una miscela di O 2 e CO 2 di bicarbonato di soluzioni basate è critica. Ipossia prevenzione e iperossia è importante mentre un misuratore di portata può essere utilizzato. Abbiamo anche usato in certe situazioni un dispositivo specifico che misura dell&#39;ossigeno disciolto nei liquidi balneari tessuto. In certe condizioni in cui i muscoli sono more soggetto a danni, la manipolazione del muscolo isolato in una Ca 2 +-free soluzione prima di montare il trasduttore di forza aiuterà a rilassare il muscolo e ridurre al minimo il danno muscolare prima dell&#39;esperimento. Farmaci come 2,3 - butanedione monoxime (BDM) o N-benzil-p-toluene sulfonamide (BTS) può anche essere aggiunto durante la dissezione per minimizzare i danni, in particolare se fasci muscolari o strisce muscolari vengono preparati, che è comune per studio contrattilità del diaframma 37, 38. Durante la registrazione, preparazioni danneggiati si generano meno forza e sono aumentati rumore basale e fluttuazioni (vedere inserto di figura 2), ed i risultati di questi muscoli possono essere esclusi dal dataset analisi, a meno che non è parte dell&#39;esperimento per analizzare le risposte di danneggiato muscoli. Un altro punto che può essere facilmente ignorato o dimenticato è che la stimolazione elettrica stessa può essere una fonte di tossicità per i muscoli. Si consiglia di pulire periodicamente gli elettrodi e la Chamber con una soluzione di ipoclorito 1% per rimuovere eventuali contaminanti, detriti proteine, ossidazione accumulo ecc speciali precauzioni devono anche quando si regola la lunghezza del muscolo montato. È importante allungare a piccoli passi per evitare sopra-stiramento come l&#39;allineamento ottimale di filamenti sottili e spessi è fondamentale per un efficiente funzionamento cross-ponte 39. Inoltre, si raccomanda che la posizione relativa dei fasci muscolari montati gli elettrodi di stimolazione di campo viene mantenuto costante e allineate tra tutti i canali per garantire che la stessa quantità di corrente elettrica viene applicata ai muscoli isolati. Nelle nostre condizioni sperimentali, muscoli da giovani topi wild type rimanere stabile per oltre 12 ore e anche di più se antibiotici, 0,2% FBS, e aminoacidi vengono aggiunti alla soluzione di bagno. Con una cura e corretto controllo, questo sistema ex vivo in grado di fornire informazioni sulla correlazione tra Ca 2 + segnalando und contrazione muscolare senza la complessità del sistema vascolare, endocrino e il sistema nervoso. Ad esempio, la perdita di dipendenza extracellulare di Ca 2 + è una firma di età compresa tra muscolo scheletrico, mentre SR Ca 2 + rilascio disfunzione produce generalmente minore forza contrattile e veloce profilo faticoso. Come exampled qui, aspetto di alternanza meccaniche indica SR instabile, che a nostro avviso non si limita a tric-a - / - i muscoli, ma anche in altri muscoli scheletrici in condizioni patologiche che generano SR Ca 2 + sovraccarico e instabilità. Questa configurazione consente l&#39;accesso diretto del muscolo per varie manipolazioni fisiologici e farmacologici.

Muscoli scheletrici allegare ossa dello scheletro e generano forze contrattili sotto il controllo del sistema nervoso centrale. Eccitazione-contrazione (ECC) si riferisce al processo di conversione di uno stimolo elettrico ad una risposta meccanica. Ca 2 + segnalazione è un componente essenziale della funzione contrattile nel muscolo scheletrico. Efficace Ca 2 + mobilitazione dal reticolo sarcoplasmatico (SR) è un componente importante di ECC in cellule muscolari 1, 2, e cambiamenti intracellulare Ca 2 + segnalazione alla base della disfunzione contrattile corrispondente in una serie di malattie muscolari 3-5. Corretta valutazione della contrattilità muscolare è essenziale e gratuito per Ca 2 + imaging e altri test per acquisire conoscenze in funzione dei muscoli scheletrici, non solo a livello contrattile, ma anche a livello cinetico. Forza e velocità possono anche essere ottenuti per informare l&#39;importante proprietà dimuscolo potenza e lo stato del processo ECC in diverse condizioni fisiologiche e fisiopatologiche. Questo campo fecondo di ricerca ha una storia molto ricca e molte teorie di contrazione muscolare è apparso più di due millenni 6. Di ricerca del muscolo moderna comincia probabilmente in 1674-1682 con l&#39;osservazione microscopica di cross-striature e le miofibrille nelle fibre muscolari di Leeuwenhoek 6. Quasi un secolo dopo, Luigi Galvani ha osservato che muscolo si contrae rana vigorosamente quando il suo nervo è toccato con bisturi durante una scarica di scintille da una macchina elettrica distante 7-9. Contrazione potrebbe anche essere prodotto collegando il nervo gamba al muscolo attraverso un conduttore metallico. I dettagli del complesso meccanismo di segnalazione elettrica sostenuti da Galvani finalmente sono stati formulati da Hodgkin, Huxley e Katz nella loro famosa equazione 10, 11 che divenne il fondamento di elettrofisiologia. Le osservazioni notevoli di Ringer sugli effetti di Ca 2 + extracellulare sulla contrattilità del cuore rana e nei muscoli scheletrici 12-15 rappresentano il primo passo importante nel riconoscimento di Ca 2 + come un regolatore chiave della contrattilità muscolare 16, 17. Dal 1980 ad oggi una raffica di scoperte nel campo contrattilità muscolare è stato realizzato a causa dell&#39;introduzione della contrattilità muscolare e affaticabilità protocolli in murine muscoli scheletrici 18. Jones e Edwards sono stati i primi a suggerire che la fatica a bassa frequenza intermittente (indotta da esercizio fisico riduzione della forza) 19 è stato associato con modifiche ai macchinari ECC e non l&#39;apparato contrattile. Alla fine del 1980 e all&#39;inizio del 1990, Kolkeck et al 20, Kolbeck e Nosek 21, e Reid 22 sono stati utilizzando muscolo diaframma da modelli di roditori per studiare gli effetti della teofillina, cortiosterone, e dei radicali liberi sulla contrattilità del muscolo scheletrico, mentre Brooks e Faulkner sono stati i primi a riferire sulle misure di forza ripetute e misure di potenza in rapida-e-lenti i muscoli di topi 22. Inoltre, Lannegren, Westerblad, Agnello, e Westerblad stati i primi a collegare direttamente la contrattilità ex vivo con intracellulare di Ca 2 + regolamento e ha iniziato a mettere in discussione il ruolo di acidosi nella fatica muscolare 23, 24. I nostri laboratori hanno contribuito in modo significativo fin dai primi anni del 2000 verso la comprensione di nuovi geni con modulante e ruoli di regolamentazione sul muscolo ECC con ruoli critici nella contrattilità muscolare, affaticabilità, e l&#39;invecchiamento utilizzando una combinazione di studi intatti topo muscolari contrattilità, intracellulare Ca 2 monitoraggio + in fibre muscolari intatte e dalla pelle e genetico-molecolari manipolazioni 3-5, 25-29. Qui dettagliato protocollo di sperimentazione per la misurazione della contrattilità murini soleo isolate e estensore lungo delle dita (EDL muscoli), che corrispondono ad una prevalentemente lenta ossidativo (tipo I e fibre muscolari IIa) e un muscolo prevalentemente fast-glyocolytic (tipo IIb e fibre muscolari IIx) con spiccate proprietà contrattili. In questo protocollo, intatte muscolotendinee complessi sono stati isolati e immersi in un sistema PowerLab ADI Radnotti camera fornito sia con ossigeno puro o una miscela di ossigeno (95%) e CO 2 (5%). Forze contrattili sono stati generati da stimoli elettrici da uno stimolatore Erba e rilevato utilizzando un trasduttore di forza che è stato integrato con un sistema ADI PowerLab/400, consentendo la personalizzazione delle routine macro per controllare l&#39;acquisizione, la raccolta, digitalizzazione e archiviazione dei dati. Questa configurazione può misurare la forza muscolare, potenza muscolare, nonché il rapporto di forza rispetto a frequenza, affaticamento muscolare, recupero da affaticamento muscolare, velocità e complessivi proprietà cinetiche di contrazione muscolare. Inoltre, gli effetti dei farmaci sulla contrazione muscolare può essere monitorata attraverso questi esperimenti. </p&gt; I vantaggi di questo metodo consistevano nel rimuovere i componenti neuronali e vascolari dal muscolo scheletrico, consentendo la valutazione diretta delle proprietà intrinseche di contrazione muscolare. Inoltre, saggi contrattilità ex vivo consentire la manipolazione del mezzo extracellulare che circonda i muscoli isolati, che consente l&#39;uso di manipolazioni farmacologiche dei vari canali ionici permeazione e trasportatori per definire i loro ruoli fisiologici per la funzione del muscolo scheletrico. Questo sistema ex vivo ci ha permesso di scoprire recentemente un comportamento distinto alternan in taluni preparati muscolari mutanti, che erano legate ad una alterata intracellulare di Ca 2 + la gestione delle proprietà 4. Alternanza sono definiti come episodi scoppio fluttuanti di forza contrattile durante la fase di declino del profilo faticosa. Durante questi eventi forze contrattili momentaneamente aumentare di sopra del suo livello precedente di forza durante la stimolazione faticoso, forse perché più o Ca 2 + è stato rilasciato o la macchina contrattile è diventato più sensibile alle Ca 2 + 30. Trattamento con acido ciclopiazonico (CPA), un bloccante reversibile-sarcoplasmatico reticolo endoplasmatico calcio ATPasi (SERCA), caffeina, un agonista del canale rianodina (RyR) e faticoso stimolazioni ripetute possono indurre alternans meccaniche 4, suggerendo che alternans sono direttamente collegate modulazione del processo di accoppiamento CE. Dimostrazione del metodo per indurre e registrare in alternanza meccanici in configurazione contrattilità in vitro serve come esempio per mostrare i parametri diversificati sperimentali che potrebbero essere ottenuti con questo sistema o simili, sulla base di interessi di ricerca individuali. Questo metodo può essere di interesse per i ricercatori che studiano la fisiologia muscolare. Configurazione simile può essere utilizzato anche per isolati complessi muscle-tendon/ligament scheletrici di altrisedi anatomiche, nonché per singole fibre muscolari e strisce.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> 2-APB </td><td> Tocris </td><td> 1224 </td><td> Bloccante di un certo numero di Ca 2 + canali di ingresso comprese SOC e TRP ecc </td></tr><tr><td> SKF96365 </td><td> Sigma </td><td> SKF-96365 </td><td> Bloccante di un certo numero di canali di ingresso Ca 2 + compresa SOC e recettore-mediata Ca 2 + entrata ecc </td></tr><tr><td> BTP-2 </td><td> Millipore </td><td> 203890-5MG </td><td> Relativamente specifico SOC bloccante </td></tr><tr><td> CPA </td><td> Sigma </td><td> C1530 </td><td> Reversibile SERCA bloccante </td></tr><tr><td> caffeina </td><td> Sigma </td><td> C0750 </td><td> Veloce azione agonista RyR </td></tr><tr><td> Radnoti Quattro Unità tessuti per sistemi e organi Bath </td><td> Radnoti </td><td> 159920 </td><td></td></tr><tr><td> Combinazione di sostegno del tessuto / elettrodo stimolante </td><td> Radnoti </td><td> 160151 </td><td> Verticale Zig Zag tipo con il supporto del tessuto </td></tr><tr><td> Quad Ponte Amp </td><td> ADInstruments </td><td> FE224 </td><td></td></tr><tr><td> PowerLab/400 </td><td> ADInstruments </td><td></td><td> Questo prodotto non è più disponibile. Scegliere altre versioni del sistema di acquisizione dati. </td></tr><tr><td> Trasduttori di forza (5 mg - 25 g) </td><td> ADInstruments </td><td> MLT0201/RAD </td><td></td></tr><tr><td> Grafico v4.02 </td><td> ADInstruments </td&gt; <td></td><td> LabChart 7.3 è l&#39;ultima versione del software grafico. </td></tr><tr><td> S8800 doppia Pulse Digital Stimolatore </td><td> GRASS TECNOLOGIE </td><td></td><td> Questo prodotto non è più disponibile. S88X doppia uscita Piazza impulso stimolatore è uno stimolatore più recente. </td></tr><tr><td> RF trasformatore di isolamento Unità </td><td> GRASS TECNOLOGIE </td><td> Modello 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

Qui descritto è un metodo per misurare la contrattilità dei muscoli scheletrici isolati. Parametri come forza muscolare, potenza muscolare, cinetica contrattili, affaticabilità, e il recupero dopo la fatica può essere ottenuta per valutare aspetti specifici della eccitazione-contrazione (ECC) di processo quali eccitabilità, contrattilità macchinari e Ca 2 + capacità di movimentazione. Questo metodo elimina la fornitura nervoso e sangue e si concentra sul muscolo scheletrico isolato stessa. Abbiamo regolarmente usare questo metodo per identificare i componenti genetiche che alterano la proprietà contrattile del muscolo scheletrico se modulanti Ca 2 + vie di segnalazione. Qui, descriviamo un fenotipo appena identificato muscolo scheletrico, cioè, alternans meccanico, come esempio di varie informazioni e ricco che può essere ottenuto usando la prova in vitro contrattilità muscolare. La combinazione di questo test con saggi su cellule singole, approcci genetiche e biochimichesaggi Stry in grado di fornire importanti informazioni sui meccanismi di ECC nei muscoli scheletrici.

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

Descriviamo un metodo per misurare direttamente la forza muscolare, potenza muscolare, cinetica contrattili e affaticabilità dei muscoli scheletrici isolati in un<em&gt; In vitro</em&gt; Sistema tramite stimolazione del campo. Informazioni preziose su Ca<sup&gt; 2 +</sup&gt; Proprietà di manipolazione e macchinari contrattile del muscolo può essere ottenuta utilizzando diversi protocolli stimolanti.

Vivo Valutazione Ex della contrattilità, affaticabilità e alternanza in isolati muscoli scheletrici

 
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

Nanyang Technological University

Research

JoVE Journal

Biology

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

23.7K Views.  UMDNJ-Robert Wood Johnson Medical School. Descriviamo un metodo per misurare direttamente la forza muscolare, potenza muscolare, cinetica contrattili e affaticabilità dei muscoli scheletrici isolati in un In vitro Sistema tramite stimolazione del campo. Informazioni preziose su Ca 2 + Proprietà di manipolazione e macchinari contrattile del muscolo può essere ottenuta utilizzando diversi protocolli stimolanti.

Video: 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 micro-circolazione di misura nel muscolo scheletrico da Intra-vitale Microscopia

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

Ricerca

Biologia

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.

Transfection DNA dei mammiferi muscoli scheletrici con In Vivo Elettroporazione

 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.

Il Ex vivo Preparazione isolato microvasi scheletrico per le Indagini di reattività vascolare

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.

Isometrica ed eccentrica valutazione della forza dei muscoli scheletrici Isolato da modelli murini di distrofie muscolari

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.

Valutazione del calcio Sparks in intatte fibre muscolari scheletriche

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.

Misure contrattilità isolati su muscoli papillari per le inchieste sui Cardiac inotropo nei topi

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.

La proteina C-FOS immunoistologiche Detection: uno strumento utile come indicatore di percorsi centrali coinvolti nelle risposte fisiologiche specifiche<em&gt; In Vivo</em&gt; e<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.

Misurare la rigidità<em&gt; Ex Vivo</em&gt; Mouse aorte Utilizzando microscopia a forza atomica

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.

La tecnica del rene perfusi mouse isolato

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.

Misurazione dell'assorbimento di glucosio stimolato da insulina e contrazione in muscolo scheletrico maturo isolato e incubato da topi

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 ...