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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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, ?…
Representative Results
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…
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Sprague Dawley Rat | Charles River | Strain 400 | |
| Chronic contractile activity unit | Home-made | n/a | |
| CCA unit protective box (3.5 x 3.5 x 2.5 cm) | Home-made | n/a | Box should be made of opaque material or covered in an opague tape |
| Coin lithium ion batteries (3V) | Panasonic | CR2016 | |
| Medwire | Leico Industries | 316SS7/44T | |
| Solder pin (socket) | Digi-Key | ED6218-ND | |
| Zonas porous tape | Johnson & Johnson | 5104 | |
| Suture silk (Size 5) | Ethicon | 640G | |
| Suture silk (Size 6) | Ethicon | 706G | |
| Curved blunt scissor (11.5 cm Length) | F.S.T. | 14075-11 | |
| Curved blunt scissor (15 cm Length) | F.S.T. | 14111-15 | |
| Delicate haemostatic forceps (16 cm Length) | Lawton | 06-0230 | |
| Scalpel | Feather | 3 | |
| Curved forceps | F.S.T. | 11052-10 | |
| Stainless-steel rod (30 cm; 7mm diameter) | Home-made | n/a | Rod should have 5 mm slit in one end to hold the wire for tunneling under the skin |
| Clip applying forceps | KLS Martin | 20-916-12 | |
| Staples (clips) | Bbraun | BN507R | |
| Metal hooks/retractor | Home-made | n/a | |
| Povidone-iodine (500 mL) | Rougier | #NPN00172944 | |
| Ampicillin sodium | Novopharm | #DIN00872644 | |
| Metacam | Boehringer | #DIN02240463 | |
| Digital multimeter (voltmeter) | Soar Corporation | ME-501 | |
| LED digital stroboscope | Lutron Electronic Enterprise | DT-2269 |
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Citazione di questo articolo
Kim, Y., Memme, J. M., Hood, D. A. Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations. J. Vis. Exp. (131), e56827, doi:10.3791/56827 (2018).