Изменения в мышцах конечностей сократительной и пассивные механические свойства имеют важное значение биомаркеров заболевания мышц. Эта рукопись описывает физиологические тесты для измерения этих свойств в мышиной мышцы пальцев разгибателей и передней большеберцовой мышцы.
Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.
В этом протоколе, мы проиллюстрировали физиологические тесты для измерения сократительной и пассивных свойств EDL мышцы и сократительные свойства мышц TA. Одной из основных проблем в мышцах исследования физиологии оксигенации целевой мышцы. Для больших мышц (например, мышцы TA), в местах подхода является предпочтительным, поскольку диффузия кислорода из буфера Рингера не может добраться до центра мышцы в анализе пробирке. На месте подход не нарушает нормального кровоснабжения и гипоксии связанного искусственных эффектов можно избежать. EDL мышцы является одним из наиболее часто используемых мышц в исследовании физиологии. Адекватной оксигенации всей мышцы может быть достигнуто в пробирке в системе из-за малого размера мышц. Кроме того, в системе пробирке обеспечивает закрытую среду для управления концентрацией ионов (Ca 2 +, Na + и K +) и химическойКлиентские лицензии (АТФ и глюкозы), которые необходимы для оптимальной генерации силы мышц. Это дает прекрасную возможность для изучения влияния этих переменных на производстве силы.
Точное измерение сократительной и пассивные свойства мышц конечностей имеет решающее значение для изучения функции скелетных мышц. Характерные изменения этих свойств часто рассматриваются как признаки различных заболеваний мышц. Изменения в этих параметрах являются также важными показателями для определения экспериментальная терапия является эффективной или нет.
The authors have nothing to disclose.
Эта работа была поддержана грантами от Национального института здоровья (AR-49419, DD), мышечная дистрофия ассоциации (DD) и NIH грант подготовки T90DK70105 (CH).
Material | Manufacturer | Specifications and comments | |
Tissue-organ bath | Radnoti LLC, CA, USA | Water-jacket tissue bath (Cat #158351-LL), Oxygen disperser tube (Cat #160192), Luer valve (Cat#120722) | |
Circulating water bath | Fisher Scientific, Waltham, MA, USA | ||
Gas mix | Airgas National, Charlotte, NC, USA | 95% O2 and 5% CO2 | |
In vitro muscle function assay apparatus | Aurora Scientific, Aurora, ON, Canada | The system consists of a stimulator (Model# 701A), a dual-mode lever system (Model#300C or 305C), a signal interface (Model # 604B) and a test apparatus (Model# 800A) to vertically mount tissue organ bath | |
In vitro muscle function assay software | Dynamic muscle control (DMC) software and dynamic muscle control data analysis (DMA) software | ||
Mouse anesthesia cocktail mixed in 0.9% NaCl | Refer to the institutional guidelines | Ketamine (25 mg/ml), xylazine (2.5 mg/ml) and acepromazine (0.5 mg/ml). Throughout the surgical procedure, a supplement of 10 % of the initial dose may be needed to keep animal under anesthesia. | |
Sylgard | World Precision Instrument | Cat#SYLG184 | |
A custom-made Plexiglas dissection board | In house designed | Refer to Figure 1 | |
Heating lamp | Tensor Lighting Company, Boston, MA, USA | 15 Watt lamp to keep the mouse warm during dissection | |
Ringer’s Buffer | Chemicals are purchased from Fisher Scientific, Waltham, MA, USA | Composition in mM: 1.2 NaH2PO4 (Cat#S369) , 1 MgSO4 (Cat# M63), 4.83 KCl (Cat# P217), 137 NaCl (Cat# 217), 24 NaHCO3 (Cat# S233), 2 CaCl2 (Cat #C79) and 10 glucose (Cat# D16). Dissolve chemicals individually and mix in the order listed above. Store at 4 °C. | |
Stereo dissecting microscope | Nikon, Melville, NY, USA | ||
Dissection tools | Fine Science Tools, Foster City, CA, USA | Coarse forceps, coarse scissors, fine forceps (Straight and 45 ° angle) | |
Braided silk suture #4-0 | SofSilk USSC Sutures, Norwalk, CT, USA | Cat # SP116 | |
A custom-made stainless steel hook | Small Parts, Inc. | 2” long S/S 304V (0.18” diameter) for force transducer 305C or 2.5” long S/S 304V (0.012” diameter) for transducer 300C (Cat# ASTM A313) | |
In situ muscle function assay system | Aurora Scientific, Aurora, ON, Canada | The system (809B, in situ mouse apparatus) consist of a stimulator (Model# 701B), a dual-mode lever system (Model# 305C), a signal interface (Model# 604A) and a thermo controlled footplate apparatus (Model# 809A) | |
In vitro muscle function assay software | Aurora Scientific, Aurora, ON, Canada | Dynamic muscle control (DMC) software and dynamic muscle control data analysis (DMA) software | |
A custom-made TA assay animal platform | In house designed | Refer to Figure 2 | |
A custom-made stainless steel hook | Small Parts, Inc. | Cat# ASTM A313 | 0.5” long S/S 304V (0.18” diameter) |
Custom-made 25G platinum electrodes | Chalgren Enterprises, Gilroy,CA | Solder two 0.016” thick platinum wires to two 24G electric wires |
Table 1. Materials and equipment.