Name: induction of acute skeletal muscle regeneration by cardiotoxin injection
Uploaded: 2017-01-01T13:00:00
Description: Watch this Scientific Journal Video about Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection at JoVE.com

We thank the Animal House and the Integrated Microscopy Facilities of IGB-CNR. This work has benefited from research funding from the European Community's Seventh Framework Programme in the project ENDOSTEM (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue, grant agreement number 241440), the Italian Ministry of Education-University-Research (MIUR-PRIN2 010-2011) to G.M. and S.B. and PON Cluster IRMI to G.M., and the CARIPLO foundation to G.M. and S.B.

所有实验均严格按照动物研究机构的指导方针进行，并按照有关动物实验的法律公共卫生，动物健康，营养和健康的意大利卫生部食品安全主管部门批准。颈椎脱位，程序可以根据IACUC或等值要求各不相同，从机构的机构。 1.心脏毒素注射在胫骨前肌<ol><li>通过在启动过程之前，稀释在无菌磷酸盐缓冲盐水（PBS）中或在水中的心脏毒素原液制备10μM的工作心脏毒素（CTX）的溶液中。 <str...

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这里，我们描述了协议来诱导骨骼肌（ 即，CTX的肌内注射）急性损伤。它被广泛地用作一种强大的工具来研究骨骼肌再生的动态体内 。 CTX注射诱导的肌纤维，它是由肌膜的去极化和纤维12的收缩而引起的变性，并触发，导致肌肉再生事件的级联。骨骼肌是在注射和损伤后所要求的时点解剖，根据实验的要求，并用于后续的组织学分析。解剖的肌肉可以被冻结，夹杂在冷冻保...

哺乳动物成人骨骼肌是由多核肌细胞（肌纤维）是专门为收缩fascicules群体形成。每个肌纤维是细长的合胞体，由肌膜（质膜）和含有肌原纤维，这是由定期多次组织收缩蛋白（肌动蛋白和肌球蛋白丝）所包围。在成年生活中，在休息的条件下，骨骼肌有其肌细胞核1非常低的营业额;的确，肌细胞核，这是位于肌纤维的周围，肌膜下，在细胞周期的G0期被捕并且无法增殖1,2。 骨骼肌必须重新生成以下的损伤，组织重塑的几个事件紧密相关的互相后达到稳态的特有能力。急性损伤或外伤之后，变性诱导，随后通过再生过程涉及不同的细胞群，包括肌肉细胞的常住人口中，卫星细胞（SCS）。事实上，在没有任何环境刺激的，卫星细胞是处于静止状态，位于肌膜和基底层3,4之间的专门利基。以下内容的损伤或疾病，旺成为激活，增殖，迁移到受损区域，并最终分化，从而引发新形成肌纤维5。活化的SC建立与不同细胞群，主要炎症细胞，这是在创伤6-8的部位招募串扰。这种串扰允许细胞遵循稳压范例通过该分子信号驱动结构上的修改，最终导致动态平衡9。此外旺，炎症和间质细胞，血管生成过程，并重新支配的事件也都参与其中，以协调的方式采取行动，以修复此高度组织化和Specialized结构。 有在研究骨骼肌再生的各个方面，不仅要了解肌肉的生理机能，同时也提高需要全过程的深入了解治疗策略的极大兴趣。几个实验的方法是目前可用来研究不同细胞群体，信号通路，以及所涉及的分子机制的标识和功能。急性损伤的小鼠模型代表一个强有力的工具来研究这个过程的许多方面。不同常用的技术来诱导急性肌肉损伤允许研究遵循再生过程在体内 ，从非常早期阶段的过程中结束。这个协议描述从肌肉注射蛇毒来源的心脏毒素的（CTX），其诱导myolysis并触发再生过程中，最多的组织样本的分析的步骤。继CTX注射，MICE可以在不同的时间点，这取决于实验要求被牺牲，并且骨骼肌可以解剖并进行进一步的分析处理。最后，我们描述了组织切片的染色方案来执行形态学观察和基本定量分析。该协议允许对急性骨骼肌再生的体内以高度重现的方式10的研究。

<table border="0" cellpadding="0" cellspacing="0" width="957">
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Cardiotoxin from&#160;Naja mossambica mossambica</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">C9759</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe For Insulin BD Micro-Fine+ Needle 30 G X 8 mm - Da 0,3 ml</td>
			<td style="width:199px;">BD</td>
			<td style="width:119px;">324826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Tragacanth Gum</td>
			<td style="width:199px;">MP BIOMEDICALS,LLC</td>
			<td style="width:119px;">104792</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methylbutane (Isopentane)</td>
			<td>SIGMA ALDRICH</td>
			<td style="width:119px;">78-78-4.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">OCT Killik Solution For Inclusion Cryostat</td>
			<td>Bio-optica</td>
			<td>&#160;05-9801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Feather Microtome Blade S35</td>
			<td style="width:199px;">Bio-optica</td>
			<td style="width:119px;">&#160;01-S35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Glass Slide Superfrost Plus</td>
			<td style="width:199px;">Menzel-Gl&#228;ser</td>
			<td style="width:119px;">09-OPLUS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Dumon #5 Mirror Finish Forceps&#160;</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">11251-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Scissors Straight Sharp/Sharp</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">15024-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Scissors Noyes Straight</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">15012-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Fine Iris Scissors Straight Sharp/Sharp 10,5 Cm</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">14094-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Eukitt</td>
			<td style="width:199px;">Bio-optica</td>
			<td style="width:119px;">09-00100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Slide Coverslip</td>
			<td style="width:199px;">BIOSIGMA</td>
			<td style="width:119px;">VBS651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Xylene</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">214736</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol 100%</td>
			<td>sigma-Aldrich</td>
			<td>02860-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Hematoxyline</td>
			<td style="width:199px;">J.T. BAKER</td>
			<td style="width:119px;">3873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Eosin</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">HT110116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Cryostat</td>
			<td style="width:199px;">LEICA</td>
			<td style="width:119px;">CM3050 S</td>
			<td></td>
		</tr>
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induction of acute skeletal muscle regeneration by cardiotoxin injection

骨骼肌再生是发生在成人骨骼肌中响应损伤或疾病的生理过程。急性损伤诱导的骨骼肌再生是研究参与肌肉再生以及机制和不同玩家的事件一种广泛使用的，强大的模型系统。实际上，这个过程的详细知识是为了更好地理解的病理状况导致骨骼肌变性必需的，而且它在确定新的靶向治疗策略有助于。目前的工作描述了一个详细的和可重复的协议，并通过单次肌注的心脏毒素（CTX）诱导小鼠急性骨骼肌再生。 CTX属于家庭的蛇毒毒素和导致肌纤维的myolysis，最终触发再生事件。骨骼肌再生的动力学是由肌肉切片的组织学分析评价。该协议还示出了用于解剖，冷冻，并切割该胫骨前肌，以及，被广泛用于后续形态学和形态分析例程苏木曙红染色的实验程序。

H＆E染色允许在特定时间点的再生处理的形态的骨骼肌再生过程中进行评价。 图3显示了对野生型小鼠的受伤的TA肌肉进行时间过程分析。肌肉已经分离在CTX注射后3,7，15，和30天，如在图3A中图式。 H＆E染色横截面有代表性的图片显示肌肉修复的动力随着时间的推移（ 图3B-E）。在伤后第3天，肌肉的结构体系结构被完全破坏，并且两个...

Watch this Scientific Journal Video about Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection at JoVE.com

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

该原稿描述了详细的协议来诱导成年小鼠和肌肉的随后的操作，如解剖，冷冻，切割，常规染色，和肌纤维横截面积分析急性骨骼肌再生。

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

The overall goal of this procedure is to obtain a mouse model of skeletal muscle acute injury to study the regeneration process in vivo, from the very early stages to the end of the process. Mouse models of muscle acute injury is a powerful tool to investigate many aspects of the regeneration process. The main advantage of this technique is the high reproducibility of all the steps involved.To begin this procedure, apply a few drops of 70%ethanol onto the the hind limb of an anesthetized mouse. To visualize the exact location of the tibialis anterior muscle, remove the hair at the anterior part of the lower leg with a scalpel. To inject the cardiotoxin solution, draw 100 microliters of it into the syringe.Hold the syringe upright and remove the air bubbles. Then, insert the needle in the center of TA from the tendon towards the knee, following the position of the tibia bone as guidance. Perform the injection without going too deep, and avoid going beyond the muscle itself.Wait two to three seconds before pulling out the needle to prevent leakage of cardiotoxin from the muscle. Apply a few drops of 70%ethanol onto the hind limb of the euthanized animal. Cut the skin at the level of the proximal tendon, then insert one of the blades of the scissors under the skin and cut the skin in the direction of the knee.Pull up the skin to expose the muscle entirely. Carefully remove all the skin and the hair from the area of interest. After that, eliminate the fascia by slowly pinching or cutting the extreme periphery of the muscle.Once broken, gently remove the fascia with the help of the thin-tipped tweezers, avoiding damaging the underlying muscle. Visualize the distal tibialis anterior tendon above the foot, then separate the distal tibialis anterior tendon from the extensor digitorum tendon by passing the tweezers'tip below the tendon. Gently slide the tweezers below the tibialis anterior to separate it from the underlying muscle.Hold the tweezers under the muscle to keep it slightly raised. Cut the tibialis anterior tendon and gently pull it upwards until only the area below the knee is attached. Subsequently, cut the TA below the knee, following the edge of the muscle with the scissors.Place a small amount of tragacanth gum on a slice of cork. Insert the distal tendon of the TA into the tragacanth gum, and leave about 3/4ths of the muscle outside, making sure to have the muscle in a perpendicular position with respect to the cork. After that, fill an aluminum can with isopentane.Suspend the can of isopentane in a dewar containing liquid nitrogen. When isopentane reaches the proper temperature, white solid particles form at the bottom of the can. Rapidly dip the cork with the muscle into isopentane, keeping the muscle position downwards.While the specimen floats, only the reverse side of the cork should be visible in the liquid. Leave the sample in isopentane for one minute. After that, immerse the muscle in liquid nitrogen for at least two minutes.Then, wrap the specimens individually in labeled aluminum foil and immediately place them in dry ice. During the procedure, it is extremely important to maintain the correct temperature and to prevent the specimen from thawing. In this procedure, place the specimen stub, the blade, and the specimen in the cryostat chamber, allowing them to equilibrate for at least 30 minutes.Next, place a small amount of freezing compound on the specimen stub, and immerse the cork with the specimen in the OCT compound before the freezing compound solidifies. Place the blade in the blade holder. Place the specimen in its proper holder.Cut the sections at a thickness of 10 micrometers. Then, place the sections on a polarized slide. After sectioning, store the slides at 80 degrees Celsius.In a chemical hood, completely air dry the frozen slides with sections for 10 to 15 minutes at room temperature. Then, lodge the slides in a staining tray. Stain the sections for one minute with hematoxylin.After that, lay the staining trough under a fairly weak tap water jet for 10 minutes to wash out the hematoxylin. Following that, counterstain the sections with eosin solution for one minute, then rinse them with water to eliminate excessive eosin solution. Subsequently, dehydrate the samples in a graded ethanol series at 50%followed by 70%Afterward, rapidly immerse the samples in 95%ethanol, followed by two changes in 100%ethanol.Clear the samples in two changes of xylene, each for at least five minutes. To mount the slides, apply a few drops of the xylene-based mounting medium on each slide, and cover it with a cover slip. Keep the slides in the fume hood for a few hours, or overnight.Shown here is the experimental scheme of the acute skeletal muscle injury. Here are the representative pictures of hematoxylin-and eosin-stained sections of cardiotoxin-treated muscles at the indicated days after injury. A couple of necrotic fibers, which is enclosed by the black continuous line, are likely invaded by the inflammatory cells.The dashed line marks an area of the infiltrating mononucleated cells. A single immature myofiber with centrally located nucleus is indicated by the black circle. Here are the large regenerated eosinophilic myofibers, with the centrally located nuclei that are marked by the black circles.This graph shows the average of the centrally-nucleated myofiber size values in TA muscle sections. And this graph shows the myofiber cross-sectional area distribution at 6, 15, and 30 days after cardiotoxin injection. Following this procedure, skeletal muscle sections can be used to perform different histological and immunofluorescence protocols to identify specific features of the muscle tissue.After watching this video, you should have a good understanding of how to investigate different aspects of skeletal muscle regeneration process that follow acute injury.

Research

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The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

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Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

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Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

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

完整毛里丝骨骼肌的X射线衍射作为研究肌肉疾病结构基础的工具

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