نشكر ليختمن JW نصيحته في المراقبة المجراة في العضلات.

RBC غشاء تلوين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تم تصحيح خلايا الدم الحمراء الماوس من سلالة الفأر نفسه مع heparinization. </li><li style=";text-align:right;direction:rtl"> وكانت ملطخة الماوس RBC مع PKH26 صبغ (551/567 نانومتر ؛ الأحمر رابط الخلية الفلورسنت عدة ، سيغما ، الولايات المتحدة). </li><li style=";text-align:right;direction:rtl"> وجرفت المياه RBC في برنامج تلفزيوني وحضنت مع الحل 2μM PKH26 من صبغ لمدة 5 دقائق في درجة حرارة الغرفة. وكان رد الفعل توقفت بإضافة البلازما (المعطل الحرارة لمدة 1 ساعة على 65 درجة مئوية مسبقا) وحضنت لمدة 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> تم غسلها RBC الملون 5 مرات مع برنامج تلفزيوني. </li></ol> إعداد الماوس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> واستخدمت ثلاثة إلى ستة أشهر من العمر من الذكور C57BL/10 الفئران في هذه الدراسة. نحن تخدير الفئران عن طريق الحقن داخل الصفاق من بنتوباربيتال (50mg/kgBW). </li><li style=";text-align:right;direction:rtl"> ثم كانت intubated الفئران مع أنبوب البولي ايثيلين عيار 20 ، التهوية الميكانيكية ، وارتفعت درجة حرارة بلغت 37 درجة مئوية على المدفأة ورقة Kapton متصلة الاستشعار الحرارية ونظام التغذية المرتدة وحدة تحكم في درجة الحرارة. </li><li style=";text-align:right;direction:rtl"> وكان حليق الجانب البطني من الرقبة. تعرض عضلات اليمين واليسار القصي الخشائي. وقدم الدعم للعضلات من جانب صاحب الفولاذ المقاوم للصدأ وsuperfused مع محلول معقم رينغر كريبس. </li></ol> حقن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وقد تحسنت في كرات الدم الحمراء الملطخة 37 درجة مئوية ، مع المخفف رينغر كريبس. تم حقن 50ul من RBC الملون (الهيماتوكريت 12 ٪) في الماوس من الوريد القضيب. </li></ol> مراقبة مجهرية في الجسم الحي من عضلات الهيكل العظمي تابعنا جيف جورج يختمن &#39;ق مع تعديل طريقة 1 : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضعت الحيوانات على لوحة التدفئة على أعلى مرحلة الحديد المصنوعة يدويا من المجهر الكسوف - 800 نيكون. وقد استخدمت المياه غمس عدسات موضوعية لرصد البرنامج الموسع للتمنيع ، فلوري حية. لاحظنا PKH26 RBC الملون داخل الأوعية الدموية التي تتدفق من تحت ضوء مصباح الفلورسنت من عطارد. حددنا الشرايين الابتدائي ، الثانوي الشرايين والشعيرات الدموية والأوردة التي توجهاتها تدفق كرات الدم الحمراء وأبنية. </li><li style=";text-align:right;direction:rtl"> تم تركيب كاميرا SIT المكثف (هاماماتسو الضوئيات ، C2400 ، اليابان) والمختطف الإطار الفيديو لالتقاط إشارة كثافة منخفضة بمعدل الفيديو في الوقت الحقيقي (30 لقطة في الثانية). وسجلت الصور على دي في دي وملفات الفيديو. </li></ol> تحفيز العضلات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ولدت نبضة كهربائية باستخدام تحفيز الأعصاب الطرفية (Innervator 252 ، فيشر آند بيكل الرعاية الصحية ، ونيوزيلندا) ، وتسليمها الى سطح العضلات عبر المسبار مطلي. وضعت نهاية لتحقيق ناقص في الجزء القريب من العضلات ، وكان وضع نهاية زائد على الجزء الأعلى من العضلات. أعطيت التحفيز الكزاز بمعدل 50HZ لمدة 5 ثوان. لم المحفزات الكهربائية لا تسبب الضرر المباشر myofiber على مقربة من منطقة التماس. </li></ol> تحليل تدفق RBC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تم نقل الصور المسجلة فيديو دي في دي ملفات الفيديو صورة سافانت من خلال برامج المختطف الإطار (فيديو سافانت ، الولايات المتحدة). وجرى استعراض صور الفيديو في سرعة تعديلها من أجل تصور RBC ملطخة الفردية. وقد تم قياس تدفق RBC RBC عن طريق العد ، والتي طارت من خلال شرين الأولية ركز في الدقيقة الواحدة. </li></ol>

<ol>
	<li>Lichtman, J. W., Magrassi, L., Purves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+neuromuscular+junctions+over+periods+of+several+months+in+living+mice.">Visualization of neuromuscular junctions over periods of several months in living mice.</a> J Neurosci. 7, 1215-1222 (1987).</li></ol>

وجرى استعراض كافة الإجراءات المتصلة التجارب على الحيوانات والتي وافقت عليها اللجنة الفرعية لرعاية الحيوان البحوث من مستشفى ماساتشوستس العام.

نقاط هامة techinical هي كما يلي : (1) الحفاظ على الوضع الفسيولوجي للحيوان (التهوية ، ودرجة الحموضة ودرجة الحرارة حل perfusative الجسم ،) ، (2) حجم حقن RBC الملون ، والظروف (3) لمراقبة (عدسة الأمثل الاختيار ، وكثافة مضان). التطبيقات المحتملة في المستقبل هي على النحو التالي : (أ) بالاشتراك مع التدخلا?...

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<td>PKH26 dye </td>
<td>&nbsp;</td>
<td>Sigma</td>
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<td>551/567 nm; Red fluorescent cell linker kit</td>
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<td>C57BL/10 mice </td>
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<td>Three to six month old males</td>
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<td>pentobarbital </td>
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<td>anesthetic, 50mg/kgBW, i.p.</td>
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in vivo micro-circulation measurement in skeletal muscle by intra-vital microscopy

معلومات أساسية : العوامل التنظيمية والفيزيولوجيا مفصل في الجسم الحي دوران الأوعية الدقيقة ظلت لم تتضح تماما بعد طرائق عديدة ومختلفة من التصوير قد اخترعت. في حين أن العديد من المعلمات العيانية تدفق الدم تعكس سرعة تدفق ، والتغيرات في سرعة تدفق الدم وخلايا الدم الحمراء (RBC) متقلب لا تعقد العلاقة الخطية في هذه الملاحظات المجهرية. هناك تقارير عن التفاوت بين سرعة وتدفق RBC RBC ، RBC تدفق البلازما وحجم التدفق والتنوع المكاني والزماني للتنظيم التدفق في الأنسجة الطرفية في الملاحظات المجهرية ، والأساس العلمي لشرط دراسات أكثر تفصيلا في مجال تنظيم استخدام microcirculatory intravital المجهري. الطريقة : نحن تعديل طريقة جيف يختمن من الملاحظة المجهرية في الجسم الحي للعضلات القصية الخشائية الماوس. الفئران هي تخدير التهوية ، وحقنوا PKH26L fluorescently كرات الدم الحمراء ، المسمى لمراقبة مجهرية. نتائج واستنتاجات : تم الكشف عن كرات الدم الحمراء وfluorescently المسمى حاليا بشكل جيد من قبل المجهر واسعة المجال. تقلص العضلات التي حركها زيادة التحفيز الكهربائي يتسبب في تدفق RBC. والكمي للمعلمات أخرى بما في ذلك السرعة والكثافة الشعرية RBC ممكنا. التسامح مع الفئران جيدا لعملية جراحية ، وحقن من كرات الدم الحمراء الملطخة ، والمراقبة المجهرية ، والتحفيز الكهربائي. لم يلاحظ أي ضرر العضلات أو الدم السفينة ، مما يوحي بأن لدينا وسيلة نسبيا أقل الغازية وملاءمة لفترة طويلة الأجل الملاحظات.

Watch this Scientific Journal Video about In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy at JoVE.com

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

ويقدم طريقة جديدة لمراقبة تنوعا من دوران الأوعية الدقيقة. فهو يعتبر مناسبة للمراقبة على المدى الطويل ، وعلى تركيبة مع التدخلات البيولوجية pharmacophysiological أو الجزيئي.

في القياس الصغير تداول المجراة في الهيكل العظمي والعضلات التي البينية الحيوية الميكروسكوب

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

in-vivo-micro-circulation-measurement-skeletal-muscle-intra-vital

Research

JoVE Journal

Biology

11.1K Views.  Massachusetts General Hospital, and Harvard Medical School. ويقدم طريقة جديدة لمراقبة تنوعا من دوران الأوعية الدقيقة. فهو يعتبر مناسبة للمراقبة على المدى الطويل ، وعلى تركيبة مع التدخلات البيولوجية pharmacophysiological أو الجزيئي.

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

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

تتبع المسار Engraftment العضلات في الحيوانات الصغيرة من قبل في فيفو نيون التصوير

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

مضان القائم على القياس من دخول الكالسيوم المتجر الذي يعمل في الخلايا الحية: من خلية السرطان مثقف على عضلة

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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.

فيفو السابقين تقييم من تعب، وانقباض في عضلات الهيكل العظمي تناوب متفرقة

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

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.

تقييم الكالسيوم سباركس السليمة في ألياف العضلات الهيكلية

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

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

في الجسم الحي تتبع نسيلي من الخلايا الجذعية المكونة للدم والسلف تميزت خمسة البروتينات الفلورية باستخدام الميكروسكوب متحد البؤر وMultiphoton

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

في فيفو تتبع 4 الأبعاد للالجذعية المكونة للدم والسلف الكبار في خلايا الفأر قبي نخاع العظم

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

قياس ديناميات الأنابيب المجهرية بواسطة المجهر القرص الغزل في المغزل ميتوتيك القطب

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of 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.

قياس امتصاص الجلوكوز المحفز بالأنسولين والانكماش في العضلات الهيكلية الناضجة المعزولة والمحتضنة من الفئران

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 ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

في قياس الجسم الحي لوظيفة العضلات إكستنور الركبة في الفئران

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

القياس الكمي للتسلل الدهني للعضلات الهيكلية عن طريق إزالة الخلايا

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.

تسليط الضوء على المؤلف: القياس الكمي القائم على إزالة الخلايا للتسلل الدهني للعضلات الهيكلية  

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.