עבודה זו נתמכה על ידי AHA SDG 10SDG2630086 לזאו X, RO1-AR061385 למא י וללכת גרנט RC2AR05896 לBrotto מ &#39;

<ol>
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המדידה של כוח ועיפנות כויץ חשובה להערכה הכוללת של תפקוד שרירי שלד. המטרה העיקרית של בדיקה זו היא לזהות שינויים בכוח השרירים ומאפיינים מעייפים תחת מצבים פתולוגיים מסוימים, כגון sarcopenia ועייפות שרירים, ולבדוק את ההשפעה של תרופות / ריאגנטים על התכווצות שרירים. מאז כוח השר...

שרירי שלד לצרף לעצמות השלד וליצור כוחות התכווצות תחת השליטה של ​​מערכת העצבים המרכזית. צימוד עירור-התכווצות (ECC) מתייחס לתהליך של המרת גירוי חשמלי לתגובה מכאנית. Ca 2 + איתות היא מרכיב חיוני בתפקוד כויץ בשרירי שלד. יעיל Ca 2 + גיוס מreticulum sarcoplasmic (SR) הוא מרכיב חשוב לECC בתאי שריר 1, 2, ושינויים בCa 2 + איתות התאי בבסיס תפקוד כויץ המקביל במספר מחל שרירים 3-5. הערכה נכונה של התכווצות שרירים היא חיונית ומחמיא לCa 2 + הדמיה ומבחנים אחרים כדי להשיג תובנה לתפקוד שרירי שלד, ולא רק ברמת כויץ, אלא גם ברמה קינטית. כוח ומהירות גם ניתן להשיג על מנת ליידע את התכונה החשובה שלכוח שרירים ומצב תהליך ECC בתנאים פיסיולוגיים וpathophysiological שונים. תחום זה של מחקר פורה יש היסטוריה עשירה מאוד ותאוריות רבות של התכווצות שרירים, הופיעו מעל אלפיים שנה 6. מחקר השרירים מודרני כנראה מתחיל ב1674-1682 באבחנה המיקרוסקופית של-קווקווים צולבים וmyofibrils בסיבי שריר על ידי 6 יוונהוק. כמעט מאה שנה מאוחר יותר, לואיג&#39;י גלוואני הבחין כי תוקף חוזי שרירי צפרדע כשהעצב שלה הוא נגע עם אזמל במהלך פריקת ניצוץ ממכונה חשמלית רחוקה 7-9. התכווצות יכולה גם להיות מיוצרת על ידי חיבור העצב לשריר הרגל דרך מוליך מתכתי. את הפרטים של מנגנון האיתות החשמלי המורכב הטיפו גלווני סופו של דבר גובשו על ידי הודג&#39;קין, הקסלי וכץ במשוואה המפורסמת שלהם 10, 11, שהפכה לבסיס של אלקטרו. התצפיות המדהימות של ריןגר על ההשפעות של תאי Ca 2 + על ההתכווצות של לב צפרדע ושרירי שלד 12-15 מייצג את הצעד הראשון החשוב בהכרה של Ca 2 + כרגולטור מפתח של התכווצות שרירים 16, 17. משינה 1980 ועד ימינו פרץ של תגליות בתחום התכווצות השרירים מומש עקב כניסתה של התכווצות שרירים ופרוטוקולי עיפנות בשרירי שלד Murine 18. ג&#39;ונס ואדוארדס היו הראשון שטענה כי עייפות לסירוגין בתדירות נמוכה (הפחתת פעילות גופנית הנגרמת בכוח) 19 היו קשורים לשינויים במכונות ECC ולא מנגנון ההתכווצות. בשנת 1980 מאוחר ו1990 המוקדמים, Kolkeck et 20 אל, Kolbeck וNosek 21, ו 22 ריד היו באמצעות שריר סרעפת ממכרסמים כדי לחקור את ההשפעות של theophyllines, cortiosterone, ורדיקלים חופשיים בהתכווצות שרירי שלד, ואילו ברוקס וFaulknאה היו ראשון לדווח על מדידות של כוח חזר ומדידות כוח ובפה מהר שרירים איטיים מעכברים 22. בנוסף, Lannegren, Westerblad, כבש, וWesterblad היו הראשון ישירות לקשר contractility vivo לשעבר עם Ca 2 + תאי רגולציה והתחיל לחקור את תפקידו של חמצת בעייפות שרירים 23, 24. המעבדות שלנו תרמו באופן משמעותי מאז 2000 מוקדמים לקראת הבנה של גני רומן עם עצב ותפקידים הרגולטוריים על השריר ECC עם תפקידים קריטיים בהתכווצות שרירים, עיפנות, והזדקנות באמצעות שילוב של מחקרי contractility שרירי עכבר שלמים, תאיים Ca 2 + ניטור ב סיבי שריר שלם ועור ומניפולציות גנטיות מולקולרית 3-5, 25-29. כאן אנו מפורטים פרוטוקול הניסוי, למדידת התכווצות של soleus המבודד Murine וlongus digitorum פושטים (EDL) שרירים, אשר תואמים את רוב איטי חמצונים (אני סוג וסיבי שריר IIa) ובעיקר שרירים מהירים glyocolytic (הסוג IIb וסיבי שריר IIx) עם תכונות התכווצות מובחנות. בפרוטוקול זה, קומפלקסים שלמים שריר גיד היינו מבודדים ורחצו במערכת ADI PowerLab Radnotti קאמרית מסופקת גם עם חמצן טהור או בתערובת של חמצן (95%) ו-CO 2 (5%). כוחות התכווצות נוצרו על ידי גירויים חשמליים מממריץ גראס וזוהה באמצעות מתמר כוח שהיה משולב עם מערכת PowerLab/400 ADI, המאפשר התאמה אישית של שגרת מאקרו לשלוט רכישה, האיסוף, דיגיטציה, והאחסון של נתונים. התקנה זו יכולה למדוד כוח שרירים, כוח שרירים, כמו גם את יחסי כוח מול תדר, עייפות שרירים, התאוששות מעייפות שרירים, מהירות ותכונות הקינטית כוללות של התכווצות שרירים. בנוסף, השפעות של תרופות על התכווצות שרירים ניתן לנטר באמצעות ניסויים אלה. </p&gt; יתרונות של שיטה זו טמונות בהסרת רכיבים העצביים וכלי דם משריר השלד, המאפשרים הערכה ישירה של המאפיינים הפנימיים של הידבקות שרירים. בנוסף, מבחני contractility vivo לשעבר לאפשר מניפולציה של הסביבה התאית המקיפה את השרירים המבודדים, המאפשרת שימוש במניפולציות פרמקולוגיים של ערוצים שונים יון חלחול ומובילים במטרה להגדיר את תפקידיהם הפיסיולוגיים לתפקוד שרירי שלד. מערכת vivo לשעבר זה אפשרה לנו לאחרונה מגלה התנהגות מובהקת alternan בהכנות שרירי מוטציה מסוימות, שהיו קשורים לCa שינה התאי 2 + טיפול נכסים 4. Alternans מוגדרים כפרקי פרץ תנודות של כוח התכווצות בשלב הירידה של הפרופיל עייף אותו. במהלך אירועים אלה כוחות התכווצות רגע להגדיל מעל רמתו הקודמת בכוח דuring גירוי מתיש, אולי כי גם יותר Ca 2 + מתפרסם או מכונות כויץ הפכו רגישות יותר לCa 2 + 30. טיפול בחומצת cyclopiazonic (רו&quot;ח), חוסם הפיך של סידן reticulum ATPase sarcoplasmic-endoplasmic (SERCA), קפאין, אגוניסט של ryanodine ערוץ (RyR) וחזרתי לעייף גירויים יכול לגרום לכל alternans המכאני 4, מה שמראה כי alternans קשור ישירות אפנון של תהליך צימוד EC. הדגמה של השיטה כדי לגרום ולהקליט alternans מכונאי בהתקנת contractility מבחנה משמשת כדוגמה כדי להראות את הפרמטרים הניסיוניים המגוונים שיכול להיות שהושגו עם מערכת זו או דומים לאלה, המבוסס על תחומי מחקר בודדים. שיטה זו עשויה להיות עניין לחוקרים הלומדים פיזיולוגיה של שרירים. התקנה דומה יכולה לשמש גם למתחמי muscle-tendon/ligament שלד מבודדים משנימקומות אנטומיים, כמו גם לסיבים בודדים ורצועות שרירים.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות (אופציונלי) </td></tr><tr><td> 2-APB </td><td> Tocris </td><td> 1224 </td><td> חוסם של מספר Ca 2 + ערוצי כניסה כולל SOC וTRP וכו &#39; </td></tr><tr><td> SKF96365 </td><td> סיגמא </td><td> SKF-96365 </td><td> חוסם של מספר Ca 2 + ערוצי כניסה כולל SOC וקולט בתיווך Ca 2 + כניסה וכו &#39; </td></tr><tr><td> BTP-2 </td><td> Millipore </td><td> 203890-5mg </td><td> חוסם SOC יחסית ספציפי </td></tr><tr><td> רו&quot;ח </td><td> סיגמא </td><td> C1530 </td><td> חוסם SERCA הפיך </td></tr><tr><td> קפאין </td><td> סיגמא </td><td> C0750 </td><td> אגוניסט RyR פעולה המהיר </td></tr><tr><td> מערכת מיקלוש רדנוטי כבר ארבע יחידת רקמות איברי אמבטיה </td><td> מיקלוש רדנוטי כבר </td><td> 159920 </td><td></td></tr><tr><td> תמיכת רקמות שילוב / אלקטרודה מגרה </td><td> מיקלוש רדנוטי כבר </td><td> 160151 </td><td> אנכי זגזג סוג עם תמיכת רקמות </td></tr><tr><td> Quad הגשר Amp </td><td> ADInstruments </td><td> FE224 </td><td></td></tr><tr><td> PowerLab/400 </td><td> ADInstruments </td><td></td><td> מוצר זה אינו זמין עוד. בחר גרסה אחרת של מערכת איסוף הנתונים. </td></tr><tr><td> מתמר כוח (5 מ&quot;ג - 25 ז) </td><td> ADInstruments </td><td> MLT0201/RAD </td><td></td></tr><tr><td> תרשים v4.02 </td><td> ADInstruments </td&gt; <td></td><td> LabChart 7.3 היא הגרסה האחרונה של תוכנת תרשים. </td></tr><tr><td> S8800 כפול Pulse דיגיטלי ממריץ </td><td> טכנולוגיות GRASS </td><td></td><td> מוצר זה אינו זמין עוד. S88X כפול פלט כיכר דופק הגירוי הוא גירוי חדש יותר. </td></tr><tr><td> יחידת בידוד שנאי RF </td><td> טכנולוגיות GRASS </td><td> דגם 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

מתואר כאן היא שיטה למדידת התכווצות של שרירי שלד מבודדים. פרמטרים כגון כוח שריר, כוח השריר, קינטיקה התכווצות, עיפנות, והתאוששות לאחר עייפות ניתן להשיג להעריך היבטים ספציפיים של צימוד עירור הכיווץ (ECC) תהליך כגון רגישות, מכונה התכווצות וCa 2 + יכולת טיפול. שיטה זו מסירה את אספקת דם והעצבים ומתמקדת בשרירי השלד המבודדים עצמו. אנו משתמשים באופן שגרתי בשיטה זו כדי לזהות מרכיבים גנטיים המשנים את נכס ההתכווצות של שריר השלד למרות ויסות Ca 2 + מסלולי איתות. כאן, אנו מתארים פנוטיפ זיהה לאחרונה שלד שריר, כלומר, alternans מכונאי, כדוגמה למידע המגוון ועשיר שניתן להשיג באמצעות בדיקה בהתכווצות שרירי מבחנה. שילוב של assay זה עם מבחנים בודדים סלולריים, גישות גנטיות וbiochemiמבחני stry יכולים לספק תובנה חשובות למנגנונים של ECC בשרירי שלד.

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

אנו מתארים שיטה ישירה למדידת כוח שרירים, כוח שרירים, התכווצות וקינטיקה עיפנות של שרירי שלד מבודדים ב<em&gt; במבחנה</em&gt; מערכת באמצעות גירוי תחום. מידע רב ערך על Ca<sup&gt; 2 +</sup&gt; מאפייני טיפול ומכונה התכווצות של השריר ניתן להשיג באמצעות פרוטוקולים שונים המגרים את הדמיון.

הערכת Vivo לשעבר של התכווצות, ועיפנות Alternans בשרירי שלד מבודדים

 
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

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

23.6K Views.  UMDNJ-Robert Wood Johnson Medical School. אנו מתארים שיטה ישירה למדידת כוח שרירים, כוח שרירים, התכווצות וקינטיקה עיפנות של שרירי שלד מבודדים ב במבחנה מערכת באמצעות גירוי תחום. מידע רב ערך על Ca 2 + מאפייני טיפול ומכונה התכווצות של השריר ניתן להשיג באמצעות פרוטוקולים שונים המגרים את הדמיו?...

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.

במדידה Micro-vivo מחזור בשרירי השלד על ידי מיקרוסקופית תוך חיוני

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

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.

ה-DNA transfection של שרירי השלד היונקים באמצעות 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 ...

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.

לשעבר vivo הכנה מבודדת Microvessel השלד לחקירות תגובתיות כלי דם

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.

הערכת דור חיל איזומטרי והאקסצנטרית של שרירי שלד מבודד ממודלים עכבריים של ניוון שרירים

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.

הערכה של סידן ניצוצות בסיבי שריר שלד שלמים

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.

מדידות התכווצות שרירים בpapillary מבודדים לחקירת הלב Inotropy בעכברים

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.

חלבון C-FOS איתור immunohistological: כלי שימושי כסמן של מסלולים מרכזיים המעורבים בתגובות פיסיולוגיות ספציפיות<em&gt; In vivo</em&gt; ו<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.

מדידת הקשיחות של<em&gt; Ex Vivo</em&gt; עכבר Aortas באמצעות מיקרוסקופ כוח אטומי

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

מדידת ספיגת גלוקוז מגורה באינסולין ובהתכווצות בשרירי שלד בוגרים מבודדים ומדגרים מעכברים

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 ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

תפילת מיופיבר יחיד לבדיקה להערכת פונקציונליות תאי גזע שריר למבוגרים Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...