Финансирование не объявлять.

<ol>
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The authors have no conflicts of interest.

Это видео обеспечивает шаг за шагом резюме скелетной процедуры биопсии мышц, используемой в АСУ лаборатории Performance человека. Эта процедура, с небольшими модификациями, был использован для сбора около 1600 мышечные биопсии в течение последних 13 лет. Образцы мышечной биопсии предоставили ...

Чрескожная или «полуоткрытый» методика биопсии используется для получения скелетных образцы мышечной ткани от больных людей и предметов исследования. Дюшенна (1806-1875) приписывают как первый построить иглу с троакар для получения скелетной мышцы от живых субъектов через чрескожной биопсии 1. В 1960-х годах, Bergström представила чрескожной биопсии иглы подобный тому, который описан Дюшенна 2-4. Двадцать лет спустя, Эванс и др. 5 модифицировали технику, применяя всасывание через режущей троакар иглы Bergström. Эта модификация может увеличить выход ткани от 3 ​​до 5-кратного 6,7 и используется в клинических и медико-биологических исследований настроек. Этот метод имеет и будет продолжать укреплять диагноз миопатии и наше понимание структуры и функции скелетных мышц. Техника биопсии чрескожная мышцы straightforward. Если все сделано правильно, при строгом соблюдении асептики, связанные с этим риски минимальны. Процедура биопсии мышц рассматривается как исследуемого инструмента для научно-исследовательских проектов. Некоторые учебные заведения позволяют подготовку преподавателей исследователей со степенью доктора наук, с прямым врача надзора, чтобы получить биопсию мышц и другие требуют сертифицированный врач выполнять технику. Биопсия команда АСУ успешно выступал более 1600 биопсию мышц в течение последних 13 лет 8-15. Цель этого видео заключается в описании изменение Bergström иглы чрескожная мышц технику биопсии 5 получить образцы ткани скелетных мышц от латеральной широкой человеческих субъектов.

<table><tbody><tr><td>Bergstr&amp;ouml;m biopsy needle, 5 x 100 mm</td><td>Stille Surgical Inc.</td><td>119-29187-50</td><td>1 ea</td></tr><tr><td>Durasorb blue pad</td><td>Fisher</td><td>22-031-340</td><td>case of 300</td></tr><tr><td>Prep razor disposable</td><td>Moore Medical</td><td>89760</td><td>1 ea</td></tr><tr><td>Shave cream</td><td>Moore Medical</td><td>92869</td><td>1 can</td></tr><tr><td>Alcohol swab</td><td>Moore Medical</td><td>98721</td><td>box/200</td></tr><tr><td>Povidone-iodine swabsticks (3 pack)</td><td>Moore Medical</td><td>90691</td><td>pack of 50</td></tr><tr><td>Face masks</td><td>Fisher</td><td>19-039-690</td><td>case of 50</td></tr><tr><td>Sterile gloves; sz 6.5</td><td>Moore Medical</td><td>68128</td><td>box of 50 pairs</td></tr><tr><td>Sterile gloves; sz 7.0</td><td>Moore Medical</td><td>68129</td><td>box of 50 pairs</td></tr><tr><td>Sterile gloves; sz 8.5</td><td>Moore Medical</td><td>68132</td><td>box of 50 pairs</td></tr><tr><td>Fenestrated towel drape</td><td>Moore Medical</td><td>92712</td><td>case of 50</td></tr><tr><td>Lidocaine (1% HCl, w/o Epi)</td><td>Dealmed</td><td>427902</td><td>30 cc vial</td></tr><tr><td>Ethyl chloride spray</td><td>Dealmed</td><td>386020</td><td>3.5 fl. Oz. bottle</td></tr><tr><td>5 ml syringe w/ 21 G needle</td><td>Fisher</td><td>14-827-48</td><td>case of 100</td></tr><tr><td>25 G 5/8&quot; needle</td><td>Fisher</td><td>14-826AA</td><td>case of 100</td></tr><tr><td>25 G 1 1/2&quot; needle</td><td>Fisher</td><td>14-826-49</td><td>case of 100</td></tr><tr><td>Single-use scalpels w/ #11 blade</td><td>Fisher</td><td>0-8927-5B</td><td>box of 20</td></tr><tr><td>60 ml Syringe</td><td>Fisher</td><td>13-689-8</td><td>case of 40</td></tr><tr><td>~30 inch extension tubing</td><td>Fisher</td><td>14-169-7A</td><td>roll of 50 ft</td></tr><tr><td>Metal stopcock</td><td>Fisher</td><td>01-290-38</td><td>each</td></tr><tr><td>Polypropylene tubing connector w/ tapered ends, 3.2 to 5.5 mm</td><td>Fisher</td><td>15-315-28A</td><td>case of 100</td></tr><tr><td>Yellow pipette tips (1-200 &amp;micro;l)</td><td>Fisher</td><td>02-681-2</td><td>cut off ~15-18 mm of the tip</td></tr><tr><td>Topical surgical adhesive</td><td>Medline</td><td>MSC091076</td><td>case of 36 tubes</td></tr><tr><td>Topical surgical adhesive</td><td>Medline</td><td>MSC091076Z</td><td>box of 6 tubes</td></tr><tr><td>Butterfly stitches (adhesive strips) 1/2&quot; x 4&quot; (6 per pack)</td><td>Moore Medical</td><td>93050</td><td>box/300</td></tr><tr><td>Bandadge, Strip, 3/4 x 3 in.</td><td>Fisher</td><td>19-027-202</td><td>pack of 100</td></tr><tr><td>Adhesive bandage (5 m rolls)</td><td>Fisher</td><td>19-027-768</td><td>case of 36</td></tr><tr><td>1&quot; paper surgical tape</td><td>Fisher</td><td>19-027-761</td><td>case of 12</td></tr><tr><td>Sutures (4-0 reverse cutting, FS2)</td><td>Moore Medical</td><td>82926</td><td>box of 36</td></tr><tr><td>4 x 4 Sterile gauze pads</td><td>Moore Medical</td><td>66949</td><td>pkg/50</td></tr><tr><td>2 x 2 Non-sterile gauze pads</td><td>Moore Medical</td><td>37336</td><td>pkg/200</td></tr><tr><td>3 x 4 Non-adherent pads</td><td>Moore Medical</td><td>74389</td><td>bx/100</td></tr><tr><td>Autoclave bags 3.5 x 9</td><td>Moore Medical</td><td>40390</td><td>box of 200</td></tr><tr><td>Autoclave bags 5.25 x 10</td><td>Moore Medical</td><td>40391</td><td>box of 200</td></tr><tr><td>Autoclave indicator tape</td><td>Moore Medical</td><td>31608</td><td>box of 250</td></tr><tr><td>Lab coats&amp;mdash;small</td><td>Fisher</td><td>23-900-520A&amp;nbsp;</td><td>10/pk</td></tr><tr><td>Lab coats&amp;mdash;medium</td><td>Fisher</td><td>23-900-520B&amp;nbsp;</td><td>10/pk</td></tr><tr><td>Lab coats&amp;mdash;large</td><td>Fisher</td><td>23-900-520C&amp;nbsp;</td><td>10/pk</td></tr><tr><td>Germicidal Disposable Cloth Wipes</td><td>Moore Medical</td><td>62879</td><td>Can/160</td></tr></tbody></table>

human skeletal muscle biopsy procedures using the modified bergstr&#246;m technique

Чрескожная техника биопсия позволяет исследователи и клиницисты, чтобы собрать образцы ткани скелетных мышц. Методика безопасна и высоко эффективна. Это видео описывает чрескожную технику биопсии, используя модифицированную Бергстром иглу для получения образцов ткани скелетных мышц от латеральной широкой человеческих субъектов. Бергстрём иглы состоит из наружной канюли с небольшим отверстием («окно») на стороне наконечника и внутренней троакара с режущим лезвием на дистальном конце. Под местной анестезией и асептических условиях, игла продвигается в скелетных мышцах через разрез в коже, подкожной клетчатки, и фасции. Далее, всасывающий применяется к внутренней троакара, внешний троакара отведена назад, ткани скелетных мышц втягивается в окне внешней канюли всасывания, а внутренний троакара быстро закрыт, таким образом, резки или отсечения образец ткани скелетных мышц . Игла поворачивается на 90 °60, а другой надрез. Этот процесс может быть повторен еще три раза. Этот метод множественного резки, как правило, производит выборку 100-200 мг или более в здоровых субъектов и может быть сделано непосредственно перед, во время и после приступа упражнения или другого вмешательства. После пост-биопсии декорирования месте разреза, предметы, как правило, возобновить свою деятельность в повседневной жизни сразу же, и может в полной мере участвовать в энергичной физической деятельности в течение 48-72 часов. Субъекты должны избегать тяжелые упражнения сопротивления в течение 48 часов, чтобы снизить риск грыжи мышцы через разрез в фасции.

Процедура биопсии мышц, как описано выше, позволяет исследователю быстро и последовательно собрать образцы ткани скелетных мышц. Типичный выход в здоровых, спортивных субъектов составляет 200 мг или больше в один проход с 3-4 клипов. Процедура занимает 15-20 минут, большинство из которых пр...

Watch this Scientific Journal Video about Human Skeletal Muscle Biopsy Procedures Using the Modified BergstrÃ¶m Technique at JoVE.com

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Человеческого скелета Процедуры биопсии мышц с использованием модифицированного Бергстром Техника

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

human-skeletal-muscle-biopsy-procedures-using-modified-bergstrm

Research

JoVE Journal

Medicine

39.8K Views.  Appalacian State University. The purpose of this video is to present the modified Bergström skeletal muscle biopsy technique on human subjects. 

Video: Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Выделение и количественный Иммуноцитохимическая Характеристика первичных Миогенные клеток и фибробластов из человеческих скелетных мышц

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Developmental Biology

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

Рентгеновская дифракция институющих Морин скелетной мышцы в качестве инструмента для изучения структурной основы мышечной болезни

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

Изоляция, Культура, Характеристика и Дифференциация клеток-прародителей мышц человека от процедуры биопсии скелетных мышц

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Выполнение человеческих скелетных мышц ксенотрансплантатов в иммунодефицитных мышей

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

Количественная оценка распределения субклеточного гликогена в волокнах скелетных мышц с помощью просвечивающей электронной микроскопии

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Biochemistry

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Подготовка и Respirometric Оценка митохондрий, изолированных от скелетной мышечной ткани, полученные чрескожной пункционной биопсии

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

MyDoBID - новый метод эффективного определения типов мышечных волокон

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Автор в центре внимания: Расшифровка тайн типов скелетных мышечных волокон с помощью техники MyDoBID 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

Идентификация типа волокна скелетной мускулатуры человека

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Человек<em&gt; латеральной широкой</em&gt; Скелетных мышц Биопсия Используя Weil-Blakesley конхотом

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Человеческого скелета Процедуры биопсии мышц с использованием модифицированного Бергстром Техника

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