מחקר נתמך על ידי מדעי הטבע והנדסת מועצת המחקר של קנדה.

<ol>
	<li>MacIntosh, B. R., Gardiner, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttetanic+potentiation+and+skeletal+muscle+fatigue:+interactions+with+caffeine.">Posttetanic potentiation and skeletal muscle fatigue: interactions with caffeine.</a> Canadian Journal of Physiology and Pharmacology. 65, 260-268 (1987).</li><li>Dormer, G. N., Teskey, G. C., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-frequency+and+force-length+properties+in+skeletal+muscle+following+unilateral+focal+ischeaemic+insult+in+a+rat+model.">Force-frequency and force-length properties in skeletal muscle following unilateral focal ischeaemic insult in a rat model.</a> Acta. Physiol. (Oxf.). 197, 227-239 (2009).</li><li>MacIntosh, B. R., MacNaughton, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+length+dependence+of+muscle+active+force:+considerations+for+parallel+elastic+properties.">The length dependence of muscle active force: considerations for parallel elastic properties.</a> J. Appl. Physiol. 98, 1666-1673 (2005).</li><li>Tubman, L. A., MacIntosh, B. R., Rassier, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+myosin+light+chain+phosphorylation+and+twitch+potentiation+in+atrophied+skeletal+muscle.">Absence of myosin light chain phosphorylation and twitch potentiation in atrophied skeletal muscle.</a> Canadian Journal of Physiology and Pharmacology. 74, 723-728 (1996).</li><li>MacNaughton, M. B., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+length+during+repetitive+contractions+on+fatigue+in+rat+skeletal+muscle.">Impact of length during repetitive contractions on fatigue in rat skeletal muscle.</a> Pflugers. Arch. 455, 359-366 (2007).</li><li>MacNaughton, M. B., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reports+of+the+Length+Dependence+of+Fatigue+are+Greatly+Exaggerated.">Reports of the Length Dependence of Fatigue are Greatly Exaggerated.</a> J. Appl. Physiol. 101, 23-29 (2006).</li><li>Rassier, D. E., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Length-dependent+twitch+contractile+characteristics+of+skeletal+muscle.">Length-dependent twitch contractile characteristics of skeletal muscle.</a> Canadian Journal of Physiology and Pharmacology. 80, 993-1000 (2002).</li><li>Rack, P. M. H., Westbury, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+length+and+stimulus+rate+on+tension+in+the+isometric+cat+soleus+muscle.">The effects of length and stimulus rate on tension in the isometric cat soleus muscle.</a> Journal of Physiology. 204, 443-460 (1969).</li><li>Devrome, A. N., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biphasic+force-velocity+relationship+in+whole+rat+skeletal+muscle+in+situ.">The biphasic force-velocity relationship in whole rat skeletal muscle in situ.</a> J. Appl. Physiol. 102, 2294-2300 (2007).</li><li>Drzymala-Celichowska, H., Krutki, P., Celichowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summation+of+motor+unit+forces+in+rat+medial+gastrocnemius+muscle.">Summation of motor unit forces in rat medial gastrocnemius muscle.</a> J Electromyogr. Kinesiol. 20, 599-607 (2010).</li><li>Petrofsky, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+recruitment+and+firing+frequencies+of+motor+units+in+electrically+stimulated+muscles+in+the+cat.">Control of the recruitment and firing frequencies of motor units in electrically stimulated muscles in the cat.</a> Medical &#38; Biological Engineering &#38; Computers. 16, 302-308 (1978).</li><li>Bawa, P., Binder, M. D., Ruenzel, P., Henneman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+order+of+motoneurons+in+stretch+reflexes+is+highly+correlated+with+their+axonal+conduction+velocity.">Recruitment order of motoneurons in stretch reflexes is highly correlated with their axonal conduction velocity.</a> Journal of Neurophysiology. 52, 410-420 (1984).</li><li>Dormer, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental Contractil Properties of Skeletal Muscle Following a stroke in a Rat Model. Master's Thesis. , (2008).</li><li>MacNaughton, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Length dependence of Fatigue and of Repetitive Contractions. Master's Thesis. , (2005).</li></ol>

אין ניגודי האינטרסים הכריזו.

תוצאות התכווצות באיכות טובה ניתן להשיג עם טיפול בהכנה כירורגית, מאובטח גובר במכשירים ואלקטרוניקה באיכות טובה. כאשר תלמיד לומד את הניתוח הזה, כמה תלושים נפוצים כוללים: מתיחת גיד הנשה, לשבש את זרימת הדם, ודימום מוגזם. העצב חייב להיות מטופל בזהירות כדי למנוע ניזק. אתה יודע שיש לך נפגעת העצב אם כוח tetanic המקסימאלי באורך אופטימלי הוא פחות באופן משמעותי מזה שמוצג באיור 3, או אם מתח הגירוי מקסימאלי לצורך להפעיל את כל היחידות המוטוריות גדול מ 5 וולט. זה יחסית קל, כדי למנוע את כלי הדם המשרתים השריר במהלך הניתוח. הפרעה לכלים האלה יכול להתרחש כאשר המקדח שיוצב בשטח הזנב של עצם הירך. אם חור הטייס הוא לא על המשטח השטוח של עצם הירך, התרגיל עלול להחליק. כאשר זה קורה, קיימת אפשרות כי כלי popliteal לקבל משובשים. אםברכות דם סביב השרירים לאחר הגדרה, זה סימן שיש לך שבש הכלים האלה. דימום יתר יכול להתרחש גם אם וריד גדול נחתך ולא קשור. ורידים גדולים, לשים על אלה סביב הקרסול. בהכנת שרירי אתר הוא גישה יקרה ערך לחקר תכונות שרירי התכווצות. יחידות מוטוריות בודדות יכולות להיות מופעלות 10, אבל בדרך כלל את כל היחידות המוטוריות מופעלות סינכרוני. זהו חסרון ביחס להפעלה אסינכרונית הרגילה שמתרחשת על ידי גיוס יחידת מנוע מרצון, ולכן מייצג את הגבלה. עם זאת, בצד החיובי, הפעלת סינכרוני מאפשרת כימות של תגובה ממוצעת של כל היחידות המוטוריות. ישנן שתי גישות שהיו בשימוש כדי להימנע מהפעלת סינכרוני. אחת הוא להשתמש בשרוול האלקטרודה עם כמה זוגות של חוטים המגרים את הדמיון. זה מאפשר הפעלה של חלק מהיחידות המוטוריות בכל זוג,וגירוי יכול לסובב דרך הזוגות כדי להשיג הפעלה אסינכרונית. שיטה זו של הפעלה יכולה להיות משולבת עם בלוק anodal 11 כדי לנסות להפעיל את היחידות מוטוריות ברצף המתאים בהתאם לעיקרון הגודל 12. בגישה זו, כל היחידות המוטוריות מופעלות עם זוג הפרוקסימלי של אלקטרודות, ובלוק מוטל עם גירוי זרם ישיר. המשרעת של גירוי לבלוק יכולה להיות מווסתת לעכב יחידות מוטוריות שלהפעלה אינה רצויה. כנראה הבלוק משפיע האקסונים גדולים במתח הנמוך ביותר, ומשפיע בהדרגת יחידות קטנות יותר. בהכנת שרירי הגסטרוקנמיוס עכברוש אתר הוא גישה פיזיולוגית חשובה למחקר של התכווצות שרירי שלד ותכונות הביוכימיות בבריאות ובמחלה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם הציוד </th><th> חברה </th><th> מספר קטלוגים </th><th> תגובות (אופציונלי) </th></tr><tr><td> קליפרס </td><td></td><td></td><td> קוצץ לחיות מחמד באיכות טובה </td></tr><tr><td> מנורת כירורגים </td><td> Dyna-Lume </td><td></td><td> כל אחד יעשה כמה </td></tr><tr><td> Myograph </td><td></td><td></td><td> אישית בנה </td></tr><tr><td> ממריץ </td><td> דשא </td><td> S-88 </td><td> כל אחד יעשה כמה </td></tr><tr><td> מסנן מגבר מד </td><td> CWE </td><td> PM-1000 </td><td></td></tr><tr><td> Telethermometer </td><td> YSI </td><td> YSI-400 </td><td></td></tr><tr><td> פלטפורמה רובוטית </td><td> Arrick רובוטיקה </td><td> MD-2 </td><td></td></tr><tr><td> מגבר Sonometric </td><td> Sonometrics </td><td> Sonolab </td><td></td></tr><tr><td> מחשבים ואיסוף נתונים </td><td> מחשב עם NI חנקrd </td><td></td><td> אישית תוכנה (LabVIEW) </td></tr><tr><td> דוד בלוק </td><td> מעבדת אונליין </td><td> Multi-בלוק </td><td></td></tr><tr><td> שרוול עצב </td><td></td><td></td><td> Custom made </td></tr><tr><td> Microstimulator </td><td></td><td></td><td> Custom made </td></tr></tbody></table>

procedures for rat in situ skeletal muscle contractile properties

יש נסיבות רבות שבם רצוי להשיג את תגובת ההתכווצות של שריר שלד בנסיבות פיסיולוגיות: זרימת הדם תקין, כל שריר שלם, בטמפרטורת גוף. זה כולל לימוד של תגובות התכווצות כמו הגברה, גרם מדרגות ועייפות posttetanic. יתר על כן, ההשלכות של טיפול במחלה, חוסר שימוש, פציעה, אימון וסמים יכולות להיות מעניין. סרטון זה מדגים נהלים המתאימים להקמה ולהשתמש בתכשיר שריר יקר ערך זה. כדי להגדיר את ההכנה הזאת, חייבת להיות החיה מורדמת, ושרירי הגסטרוקנמיוס המדיאלי הוא ניתוח מבודד, עם המקור ללא פגע. יש להקפיד לשמור על הדם ואספקת עצב. קטע ארוך של עצב הירך נקי של רקמת חיבור, וניתק proximally. כל הסניפים של הגדם הדיסטלי שאינו מעצבב את שריר הגסטרוקנמיוס המדיאלי הם נותקו. גדם העצב הדיסטלי הוא inserטד לתוך שרוול מרופד בחוטי מגרת נירוסטה. Calcaneus הוא ניתק, משאיר את חתיכה קטנה של עצם עדיין מחוברת לגיד אכילס. גבישי Sonometric ו / או אלקטרודות לelectromyography יכולים להיות מוכנסים. Immobilization ידי בדיקות מתכת בירך ושוקה מונע תנועה של מוצא השרירים. גיד אכילס מחובר למתמר הכח והעור הרפוי הוא משך בצדדים כדי ליצור מכולה שמלאה בשמן פרפין מחומם. השמן מפיץ את חום בצורה אחידה ומצמצם את אובדן חום באידוי. מנורת חום מכוונת על השריר, והשריר והעכברוש מותרים לחמם עד 37 מעלות צלזיוס בזמן שהוא מתחמם, מתח מקסימאלי ואורך אופטימלי ניתן לקבוע. אלה הם תנאים ראשוניים חשובים לכל ניסוי בכל שריר בשלמותה. הניסוי עשוי לכלול קביעת תכונות התכווצות סטנדרטיות, כמו יחסי כוח בתדר, יחסי כוח באורך, ויחסי כוח במהירות. </p&gt; עם טיפול בבידוד ניתוחי, קיבוע של מוצא השרירים והיישור של יחידת שריר הגיד עם מתמר הכח, וניתוח נתונים נכון, מדידות איכות גבוהות ניתן להשיג עם הכנת שריר זה.

Watch this Scientific Journal Video about Procedures for Rat in situ Skeletal Muscle Contractile Properties at JoVE.com

Procedures for Rat in situ Skeletal Muscle Contractile Properties

סרטון זה מדגים את ההכנה וההליכים דרושים כדי ללמוד את תגובות ההתכווצות של שריר הכנת הגסטרוקנמיוס המדיאלי החולדה כירורגית<em&gt; באתר</em&gt;. הכנה זו מאפשרת מדידה של תכונות התכווצות שריר שלד בתנאים פיסיולוגיים. החיים הם מורדמים והשריר מופרד מרקמות בקצה הדיסטלי שלה. גיד אכילס מחובר למתמר כוח, המאפשר מדידה של תגובת ההתכווצות של השרירים בC 37 מעלות במחזור שלם.

נהלים לעכברוש<em&gt; באתר</em&gt; מאפייני התכווצות שרירי שלד

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

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Biology

Procedures for Rat in situ Skeletal Muscle Contractile Properties

28.5K Views.  University of Calgary. סרטון זה מדגים את ההכנה וההליכים דרושים כדי ללמוד את תגובות ההתכווצות של שריר הכנת הגסטרוקנמיוס המדיאלי החולדה כירורגית באתר. הכנה זו מאפשרת מדידה של תכונות התכווצות שריר שלד בתנאים פיסיולוגיים. החיים הם מורדמים והשריר מופרד מרקמות בקצה הדיסטלי שלה. גי...

Video: Procedures for Rat in situ Skeletal Muscle Contractile Properties

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

למבוגרים עובריים שרירי השלד Microexplant התרבות בידוד בתאי גזע שרירי השלד

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

בידוד מיטוכונדריאלי של שריר השלד

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

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

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

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

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

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, ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

פגיעת הליזר הביא על שריר שלד עוברי דג זברה

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

זיהוי וניתוח של נזק לדנ&quot;א בשרירי שלד עכבר<em&gt; באתר</em&gt; שימוש בשיטת TUNEL

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

בידוד מיו-בריאלס מבריות מביופסיות שריר השלד וקביעת תפקוד מתכווץ עם מתמר כוח רזולוציה ננו-ניוטון

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

זיהוי, בידוד ואפיון של אבות פיברו-אדיפוגניים (FAPs) ואבעות מיוגניים (חברי פרלמנט) בשריר השלד בחולדה

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

הכנת הומוגנטים של שרירי שלד חולדה למדידות חנקות וניטריט

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...

כימות חדירת שומן בשרירי השלד באמצעות דה-צלולריזציה

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

מחבר זרקור: כימות מבוסס דה-צלולריזציה של חדירת שומן בשרירי השלד  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.