Name: application of chronic stimulation to study contractile activity-induced rat skeletal muscle phenotypic adaptations
Uploaded: 2018-01-25T13:00:00.000+00:00
Duration: 9 min 50 s
Description: Watch this Scientific Journal Video about Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations at JoVE.com

我们感谢利亚姆泰伦的专家阅读手稿。这项工作得到了加拿大自然科学和工程研究委员会 (NSERC) 对检察官的资助。检察官胡德也是一个加拿大研究椅子在细胞生理学的持有者。

所有与动物有关的程序都经过约克大学动物保护委员会的审查和批准。在抵达约克大学的动物设施, 所有的老鼠被给予至少五天, 适应到他们的环境之前, 在手术过程中, 与食物提供的ad 随意。虽然此协议已被应用于其他物种15,17,22, 目前的论文建立在 Pette 和同事们的开创性工作23上, 并着重于大鼠模型, 特别是。
1. 慢性收缩活动单位的制备
<ol><li>使用电压表, 检查硬币锂电池的潜力。 注: 每个电池的电位应为3.0 ± 0.10 v</li>
	<li>将两个或三电池插入单元的插槽中, 使总电位为 6-9 v 注意: 这是由研究人员斟酌决定在整个实验过程中保持多少潜能 (6 或9伏)。根据研究的设计和预期的刺激强度, 可以使用2或3电池。</li>
	<li>通过脉冲指示器验证该单元是否正常工作, 将该单元暴露为便携式红外线闪光灯发出的1脉冲。</li>
</ol>2. 慢性收缩活动的手术方法
注: 在手术前对所有手术工具进行消毒。在手术后和术后立即, 大鼠的体温由加热垫维持。在外科手术的褶皱上进行手术是可取的。外科医生应该戴上无菌的外科手套以及干净的实验室大衣。如有需要, 建议佩戴一次性口罩。
<ol><li>1-3% 异氟醚吸入氧麻醉大鼠, 由气体汽化器系统操作。确认动物是完全镇静通过检查后肢脚趾捏和观察呼吸深度和速率。将眼部润滑剂涂抹在眼睛上, 避免干燥。应用皮下注射昔 (0.5 毫克/毫升) 在2毫克/千克。 注意: 它也建议有一个多模态应用的镇痛 (如, 昔加利多卡因), 以减少任何疼痛期间和手术后。</li>
	<li>轻轻地剃去左后肢, 以及从颈部后面的躯干, 在前肢后面和胸前。用碘和乙醇轻轻擦拭剃光区域消毒。</li>
	<li>与动物躺在它的胃, 做一个小切口 (〜0.5 厘米), 在颈部背部的中心, 剃光区域 (在肩胛骨之间的可触及区域) 使用手术刀 (No. 10 刀片)。</li>
	<li>找到腓总神经。<ol>
<li>将动物卷到右侧, 在左后肢的皮肤上做一个 2-3 厘米长的切口。靶切口区域周围的大腿上部肌肉组是地标之间的酒窝的膝盖关节和背部接近的起源的尾巴。 注意: 在改变身体位置时, 要注意不要污染第一个切口区域。</li>
		<li>使用钝尖的弯曲手术剪刀, 解剖皮下面积广泛〜 3.5-4 厘米, 分离皮肤的基础肌肉, 以使一个口袋之间打开的皮肤和潜在的肌肉。在切口的整个圆周周围打开皮肤 (~ 1.5 厘米2)。</li>
		<li>做一个小切口 (少于0.5 厘米) 对肱二头肌肌肉与外科剪刀, 确保剪刀的秘诀是直接削减通过肌肉。</li>
		<li>轻轻打开切口区域, 直到内部肌肉组和腓总神经可见 (外部肌肉组织 (即,股二头肌) 的深度约为0.5 厘米)。使用镊子, 轻轻触摸/捏的可见神经和观察的目标肌肉群 (如, TA 肌肉) 和脚趾 (可见背) 的反应, 以确认腓总神经是孤立的。 注意: 这一步应该采取极端谨慎的做法, 以避免切割或损害神经。</li>
		<li>用金属器打开窗口, 使窗口的大小为1.5 厘米2 , 而腓神经位于窗口的中心。使用连接到表面 (或手术板) 的带和/或橡皮筋的小金属钩 (图 2A)。</li>
	</ol></li>
	<li>把电线连接到神经的两侧。<ol>
<li>准备〜 50-60 厘米聚四氟乙烯 (PTFE) 涂层精细不锈钢线, 并对折它一半。 注意: 在手术前, 在紫外线照射下暴露 PTFA 涂层的导线可能会有帮助。</li>
		<li>将铁丝的折叠部分钩入30厘米长的不锈钢棒的狭缝中。通过杆, 连同导线, 从后肢的开放口袋的皮下向小切口区域在后面的颈部, 在 L 形模式上的腿和沿背部的中心。</li>
		<li>在后肢找到导线的两端。因为所有的电线都是聚四氟乙烯绝缘的, 所以要剥线。使用手术刀, 小心地剥去铁丝的两端1.5 厘米。如果电线磨损, 把它们切掉, 再重新带上。包裹剥线结束周围一个钝21克针 (5 次), 使线圈。一旦线圈是正确的, 松开他们的针头。</li>
		<li>使用大小6-0 的外科手术丝, 确保腓总神经两侧两侧的线圈 (图 2A)。<ol>
<li>在线圈的末端做一个结, 并且缝合它在神经的左手边。确保线圈是从神经的 1.5-2.5 毫米。</li>
			<li>为了保证线圈的安全, 在线圈上应用两个或三条附加的缝合线。</li>
			<li>在神经的右手边重复这些步骤。</li>
		</ol>
</li>
		<li>应用 2-3 滴抗生素溶液 (氨苄西林生理盐水; 132 毫克/毫升), 然后仔细缝合窗口 (即,肱二头肌组织) 使用大小5-0 丝绸。</li>
	</ol></li>
	<li>松散的风剩余的电线 (约直径的食指) 和推入皮下的口袋上方的缝合切口的二头肌肌肉 (约在臀部)。</li>
	<li>再次应用 2-3 滴抗生素溶液 (生理盐水中的氨苄西林; 132 毫克/毫升)。通过装订关闭打开的皮肤。</li>
	<li>将导线 (从颈部的切口区域出来) 连接到共同评估刺激器。<ol>
<li>将针脚插座与电线连接。<ol>
<li>切断从颈部顶端的切口的导线回路, 以创建2线端 (这些导线通向腓神经两侧缝合的线圈)。</li>
			<li>使用手术刀, 剥去电线的两端0.5 厘米, 切断磨损的电线, 如果有的话。</li>
			<li>将导线的剥离部分慢慢地推入销钉插座的孔中, 并使用烙铁, 将针脚插座上的导线焊上。</li>
		</ol>
</li>
		<li>(可选) 检查电线的连接。<ol>
<li>通过鳄鱼夹子将针脚连接到大型台式刺激器单元。</li>
			<li>提供一个9伏 (0.1 毫秒, 10 赫兹) 的单脉冲, 以确认 TA 肌肉收缩和左脚 dorsiflexes。</li>
		</ol>
</li>
		<li>通过针连接线两端通过无菌纱布垫, 是〜 4 cm x 4 厘米。</li>
		<li>将针脚连接到共同国家评估刺激装置。<ol>
<li>通过在刺激盒底部的孔导线。 注: 此框为共同国家评估刺激单元的家庭制作室, 3.5 cm x 3.5 cm x 2.5 cm13。</li>
			<li>将针脚插入到共同国家评估单位的连接插座中。将共同国家评估单位轻轻放进会议厅。使用粘性的粘性, 以确保在会议厅底部的共同国家评估单位。</li>
		</ol>
</li>
		<li>使用运动胶带或多孔的外科手术带, 在剃光的躯干周围用胶带固定房间。关闭的顶部三层的录音和完成通过包装在刺激盒两侧的磁带, 以确保框 (图 2B)。</li>
	</ol></li>
	<li>检查共同国家评估是否在工作, 将该单位暴露在一个单脉冲红外光 (波长频谱 > 770 nm), 这是由便携式红外线闪光灯发射。 注: 如果共同国家评估工作正常, 研究人员将能够看到后肢肌肉 (即, TA) 是为了响应红外线而收缩的。</li>
	<li>观察鼠并监测它的温度, 直到它完全恢复知觉。将其放在单人笼内, 以防止其他动物受到任何伤害, 并在余下的研究中不留任何隧道或塑料物件, 以减轻对刺激装置或动物造成损害的危险。供应含阿莫西林的水瓶 (0.5 毫克/毫升)。</li>
	<li>1毫克/千克剂量的昔皮下每24小时的手术后, 至少持续72小时。</li>
</ol>3. 慢性收缩活动
<ol><li>手术后, 允许至少 5-7 天, 以充分恢复切口和缝合区/周围的骨骼肌肉。 注意: 在评估过程中和之后, 通过观察它们的行为 (例如,进食、饮酒和/或移动), 彻底检查每种动物的状况。此外, 通过检查在共同国家评估程序之前和之后的体重变化来确定任何严重的压力或不良影响。</li>
	<li>在共同国家评估的刺激之日, 通过便携式红外线闪光灯将刺激装置暴露在单一的红外线脉冲 (波长范围 > 770 nm) 上, 打开共同国家评估。</li>
	<li>应用3或 6 h 的10赫兹共同国家评估刺激。 注: 刺激的时间范围由研究员负责。在这些实验中, 刺激的频率从未改变过, 在我们的经验中, 通过将刺激从3延长到6小时, 在线粒体适应方面只有非常适度的增强。如果可能的话, 检查刺激和动物每 30-60 分钟。</li>
	<li>在预期的共同国家评估期之后, 通过红外线曝光 (与在单位上打开相同的程序) 关闭共同国家评估单位。</li>
	<li>如果应用多天, 请重复步骤3.2。到3.4。</li>
	<li>确定组织收集的时间。例如, 收集组织24小时后, 在最后一回合的共同国家评估 (即, 18 小时后, 最后的共同国家评估刺激的6小时), 这是在麻醉下进行的, 在共同评估的手术过程中进行。在收集所有的组织, 安乐动物通过切除心脏, 而动物仍在麻醉。</li>
</ol>

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A., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+effects+of+chronic+nerve+stimulation+upon+tibialis+anterior+muscle+in+mouse,+rat,+guinea+pig,+and+rabbit.">Species-specific effects of chronic nerve stimulation upon tibialis anterior muscle in mouse, rat, guinea pig, and rabbit.</a> Pflugers Arch. 412 (1-2), 86-92 (1988).</li><li>Ohlendieck, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+low-frequency+stimulation+on+Ca2+-regulatory+membrane+proteins+in+rabbit+fast+muscle.">Effects of chronic low-frequency stimulation on Ca2+-regulatory membrane proteins in rabbit fast muscle.</a> Pflugers Arch. 438 (5), 700-708 (1999).</li><li>Brown, M. D., Cotter, M. A., Hudlicka, O., Vrbova, G. <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+different+patterns+of+muscle+activity+on+capillary+density,+mechanical+properties+and+structure+of+slow+and+fast+rabbit+muscles.">The effects of different patterns of muscle activity on capillary density, mechanical properties and structure of slow and fast rabbit muscles.</a> Pflugers Arch. 361 (3), 241-250 (1976).</li><li>Skorjanc, D., Traub, I., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identical+responses+of+fast+muscle+to+sustained+activity+by+low-frequency+stimulation+in+young+and+aging+rats.">Identical responses of fast muscle to sustained activity by low-frequency stimulation in young and aging rats.</a> J Appl Physiol. 85 (2), 437-441 (1998).</li><li>Kim, Y., Triolo, M., Hood, D. A. <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+Aging+and+Exercise+on+Mitochondrial+Quality+Control+in+Skeletal+Muscle.">Impact of Aging and Exercise on Mitochondrial Quality Control in Skeletal Muscle.</a> Oxid Med Cell Longev. 2017, 3165396 (2017).</li><li>Callewaert, L., Puers, B., Sansen, W., Jarvis, J. C., Salmons, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+implantable+device+for+investigating+the+adaptive+response+of+skeletal+muscle+to+chronic+electrical+stimulation.">Programmable implantable device for investigating the adaptive response of skeletal muscle to chronic electrical stimulation.</a> Med Biol Eng Comput. 29 (5), 548-553 (1991).</li><li>Kern, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+counteracts+muscle+decline+in+seniors.">Electrical stimulation counteracts muscle decline in seniors.</a> Front Aging Neurosci. 6, 189 (2014).</li><li>Zampieri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+exercise+in+aging+human+skeletal+muscle+increases+mitochondrial+calcium+uniporter+expression+levels+and+affects+mitochondria+dynamics.">Physical exercise in aging human skeletal muscle increases mitochondrial calcium uniporter expression levels and affects mitochondria dynamics.</a> Physiol Rep. 4 (24), (2016).</li></ol>

作者没有什么可透露的。

慢性收缩活动 (共同国家评估) 模型的运动, 通过低频肌肉刺激在体内, 是一个优秀的模型研究肌肉表型适应锻炼13,24,25,26. 如前面的研究中所示,20,27, 共同国家评估是一个有效的工具, 研究人员可以控制训练量和频率 (即时间和天数), 并调查各种?...

骨骼肌肉可以适应运动训练, 通过改变其生物和物理结构1。耐力训练带来的主要改变之一是线粒体生物, 可以通过增加线粒体成分的表达 (例如,细胞色素 c 氧化酶 [COX] 亚基) 来评估, 以及表达转录因子, PGC-1α2。越来越多的研究表明, 许多其他因素, 包括线粒体翻转和 mitophagy, 也是重要的肌肉适应。然而, 急性或慢性运动在骨骼肌中调节这些过程的机制还不清楚。
为了描述调节运动性肌肉适应的途径, 各种运动模式在啮齿类动物研究中普遍使用, 包括跑步机、跑步轮和游泳运动。但是, 这些协议有一些限制, 因为需要 4-12 周来观察这些表型变化3,4,5。作为一种替代实验方法, 低频刺激引起的慢性收缩活动 (共同国家评估) 得到了有效的使用, 因为它可以导致肌肉适应在一个极短的时期 (即, 7 天), 其影响似乎与其他运动协议相比, 甚至更大。此外, 荷尔蒙6、温度7和神经效应8的存在可能使人们难以理解针对慢性运动的肌肉特异性反应。例如, 甲状腺激素9,10和胰岛素样生长因子 (igf-i)-111已被确定, 以调停训练诱导肌肉适应, 这也可能调节其他信号通路的骨骼肌肉.值得注意的是, 由全身因素对共同国家评估产生的影响进行了最低限度的调节, 使重点放在骨骼肌对收缩活动的直接反应上。
外部单位为共同国家评估首先介绍了由泰勒和怀特12, 并且已经开发了以修改12。简而言之, 该装置由三主要部件组成: 红外线探测器, 可通过接触红外线、脉冲发生器和脉冲指示器 (图 1) 打开和关闭。该刺激器单元的详细电路设计已在前面介绍过13。在许多审阅文章14、15、16、17中, 可以更深入地发现共同国家评估的详细和具体功能。简言之, 刺激协议的目的是激活腓总神经在低频 (即, 10 Hz), 和支配肌肉 (胫骨前 [TA] 和伸肌肌腱长长 [EDL] 肌肉) 被迫合同为预定的时间长度 (例如, 3-6 h)。随着时间的推移, 这将上述的肌肉转移到一个更有氧的表型, 显示了增加的毛细管密度18和线粒体内容19,20,21。因此, 这种方法是一个经验证的模型, 模仿一些主要的耐力训练适应大鼠骨骼肌中。
本文介绍了电极植入术的详细过程, 以使其在运动研究中得以应用。共同国家评估是研究肌肉适应时间过程的一个很好的模型, 从而为在运动训练开始后的早期和后期的各种分子和信号事件的研究提供了一个有效的工具。

<table><tbody><tr><td>Sprague Dawley Rat</td><td>Charles River</td><td>Strain 400</td><td></td></tr><tr><td>Chronic contractile activity unit</td><td>Home-made</td><td>n/a</td><td></td></tr><tr><td>CCA unit protective box (3.5 x 3.5 x 2.5 cm)</td><td>Home-made</td><td>n/a</td><td>Box should be made of opaque material or covered in an opague tape</td></tr><tr><td>Coin lithium ion batteries (3V)</td><td>Panasonic</td><td>CR2016</td><td></td></tr><tr><td>Medwire</td><td>Leico Industries</td><td>316SS7/44T</td><td></td></tr><tr><td>Solder pin (socket)</td><td>Digi-Key</td><td>ED6218-ND</td><td></td></tr><tr><td>Zonas porous tape</td><td>Johnson &amp;amp; Johnson</td><td>5104</td><td></td></tr><tr><td>Suture silk (Size 5)</td><td>Ethicon</td><td>640G</td><td></td></tr><tr><td>Suture silk (Size 6)</td><td>Ethicon</td><td>706G</td><td></td></tr><tr><td>Curved blunt scissor (11.5 cm Length)</td><td>F.S.T.</td><td>14075-11</td><td></td></tr><tr><td>Curved blunt scissor (15 cm Length)</td><td>F.S.T.</td><td>14111-15</td><td></td></tr><tr><td>Delicate haemostatic forceps (16 cm Length)</td><td>Lawton</td><td>06-0230</td><td></td></tr><tr><td>Scalpel</td><td>Feather</td><td>3</td><td></td></tr><tr><td>Curved forceps</td><td>F.S.T.</td><td>11052-10</td><td></td></tr><tr><td>Stainless-steel rod (30 cm; 7mm diameter)</td><td>Home-made</td><td>n/a</td><td>Rod should have 5 mm slit in one end to hold the wire for tunneling under the skin</td></tr><tr><td>Clip applying forceps</td><td>KLS Martin</td><td>20-916-12</td><td></td></tr><tr><td>Staples (clips)</td><td>Bbraun</td><td>BN507R</td><td></td></tr><tr><td>Metal hooks/retractor</td><td>Home-made</td><td>n/a</td><td></td></tr><tr><td>Povidone-iodine (500 mL)</td><td>Rougier</td><td>#NPN00172944</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Novopharm</td><td>#DIN00872644</td><td></td></tr><tr><td>Metacam</td><td>Boehringer</td><td>#DIN02240463</td><td></td></tr><tr><td>Digital multimeter (voltmeter)</td><td>Soar Corporation</td><td>ME-501</td><td></td></tr><tr><td>LED digital stroboscope</td><td>Lutron Electronic Enterprise</td><td>DT-2269</td><td></td></tr></tbody></table>

application of chronic stimulation to study contractile activity-induced rat skeletal muscle phenotypic adaptations

骨骼肌是一种高度适应性的组织, 因为它的生物化学和生理特性在对慢性运动的反应中有很大的改变。为了研究导致各种肌肉适应的基本机制, 在动物研究中使用了许多运动协议, 如跑步机、轮子跑步和游泳练习。然而, 这些运动模型需要很长的时间来实现肌肉适应, 这也可能受体液或神经因素的调节, 从而限制了他们的应用研究肌肉特定收缩诱导的适应。间接低频刺激 (10 Hz), 以诱导慢性收缩活动 (共同国家评估) 已被用作运动训练的替代模型, 因为它可以成功地导致肌肉线粒体适应在7天内, 独立于系统性因素。本文详细介绍了应用治疗大鼠骨骼肌的外科技术, 以便在今后的研究中得到广泛的应用。

我们已经表明, 慢性收缩活动 (共同国家评估) 是一种有效的工具, 以诱导良好的线粒体适应在骨骼肌肉。大鼠接受7天的共同国家评估 (每天6小时) 显示增强的线粒体生物在受激肌肉与刺激对侧 (控制) 后肢。线粒体生物的增加是由 PGC-1α蛋白表达增加 (图 3A), 被认为是线粒体生物的主调节器, 以及其他关键线粒体蛋白 cox I 和 cox IV 的升高,这是电子传输链...

Watch this Scientific Journal Video about Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations at JoVE.com

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

本议定书描述的使用慢性收缩活动模型的运动, 观察刺激诱导骨骼肌适应在大鼠后肢。

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 ...

application-chronic-stimulation-to-study-contractile-activity-induced

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.7K 次观看.  York University. 本议定书描述的使用慢性收缩活动模型的运动, 观察刺激诱导骨骼肌适应在大鼠后肢。

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

科研

医学

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

微型硅悬臂梁的利用率，评估细胞收缩功能<em&gt;在体外</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

生物工程

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

发育生物学

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

对大鼠的过程<em在原位</em骨骼肌收缩性能

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

生物学

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

评估人体骨骼肌微组织骨骼肌健康的功能指标

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

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.

体外评估对离体骨骼肌的收缩，疲劳性和交替“

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

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

人类神经肌肉接头光遗传学模型的工程和表征

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

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

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