最大等轴泰塔尼奇力测量大鼠的蒂比亚利斯前肌肉
Summary
运动恢复评估仍然是实验外周神经研究的基准结果衡量标准。对大鼠前肌的等分测量是评估坐骨神经缺陷重建后功能结果的宝贵工具。本文详细介绍了方法和细微差别。
Abstract
创伤性神经损伤导致大量功能丧失，段神经缺陷往往需要使用自体介置神经移植。由于其可用性和相关的供体侧发病率有限，神经再生领域的许多研究侧重于替代技术，以弥合部分神经间隙。为了研究手术或药理实验治疗方案的结果，大鼠坐骨神经模型经常被用作生物分析。大鼠模型中使用各种结果测量来确定神经再生的程度。目标肌肉的最大输出力仍然是实验疗法临床转化最相关的结果。对大鼠肌肉收缩的等分力测量以前被描述为一种可重复和有效的技术，用于评估大鼠和兔子模型中神经损伤或修复后的运动恢复。在这段视频中，我们将提供这个宝贵的程序的分步指导，以评估使用优化参数的老鼠坐骨神经缺陷模型中头肌前肌肉的功能恢复。除了手术方法和解剖常见的腹神经和头骨前肌腱外，我们将描述必要的手术前准备。将详细介绍了等轴测泰塔尼奇力测量技术。解释确定最佳肌肉长度和刺激脉冲频率，并测量最大的泰太尼肌肉收缩。
Introduction
外周神经外伤后运动功能丧失对患者生活质量和社会经济地位有重大影响。由于4年来手术技术的改善最小，这个病人群体的预后仍然很差。直接端到端无张力硬膜外修复形成黄金标准的手术重建。然而，在神经间隙扩大的情况下，自体神经移植的插位已被证明是优越的5，6。相关的捐赠网站发病率和有限的自体神经移植物的可用性已经强制需要替代技术7，8。
实验动物模型已被用来阐明外周神经再生的机制，并评估各种重建和药理治疗方案8，9的结果。大鼠坐骨神经模型是最常用的动物模型10。它们体积小，便于处理和存放。由于其最高级的神经再生潜力，干预和评估结果之间的时间减少可能导致相对较低的成本11，12。它使用的其他优点包括形态相似于人类神经纤维和大量的比较/历史研究13。虽然应谨慎对待后者，因为研究之间各种不同的结果衡量，使得很难比较结果14、15、16、17、18。
评估神经再生结果的措施范围从电生理学到组织形态学，但这些方法意味着相关性，但不一定直接测量运动功能14，15的恢复。再生神经纤维可能无法建立适当的连接，这可能导致高估功能连接的数量14，15，19，20。最好的和临床上最相关的测量，以证明正确的重新插入末端器官仍然是评估肌肉功能21，22，23。然而，为动物模型创建运动功能评估工具具有挑战性。麦地纳塞利等人首先描述了行走轨迹分析，这是自21、24、25、26、27、28实验中用于评估功能恢复的最常用方法。行走轨迹分析根据对行走大鼠21、29的爪印的测量来量化坐骨功能指数（SFI）。行走轨迹分析的主要局限性，如脚趾收缩、自动变异、打印涂抹以及与其他重新插入措施的关联性差，因此必须使用其他参数来量化功能恢复30、31。
在此前对刘易斯大鼠32和新西兰兔子33的研究中，我们验证了对头骨前部（TA）肌肉的等量泰顿力（ITF）测量，并证明了其在不同类型的神经修复34、35、36、37、38、39后肌肉恢复评估中的有效性。 TA肌肉非常适合，因为它的体积相对较大，内向由坐骨神经的腹膜分支和良好的阐明生化特性40，41，42，43。当肌肉长度（预加载力）和电气参数得到优化时，ITF 提供 32 号大鼠和 33 号兔子的侧向变异性分别为4.4%和7.5%。
本文在大鼠坐骨神经模型中提供了ITF测量的详细方案，包括对必要的手术前规划、手术方法和普通腹股神经和解剖TA肌腱的解剖的透彻描述。使用刺激强度和持续时间的预先确定值，将定义最佳肌肉长度和刺激脉冲频率。使用这四个参数，可以随后持续准确地测量 ITF。
Protocol
所有动物程序均经机构动物护理和使用委员会（IACUC A334818）批准执行。 1. 力传感器的校准 确保计算机正确连接到 USB-6009 多功能 I/O 数据采集 （DAQ） 设备，该设备应连接到力传感器。注：其他大鼠菌株和物种可能需要不同的负载细胞力转导器，因为预计更高的力为44。 将定制夹子从经过修改的手术增压器制成，连接到真空基可调杆臂上的?…
Representative Results
用于测量 ITF 测量的五个参数。这些包括肌肉张力（预加载力）、刺激强度（电压）、刺激脉冲频率、0.4毫秒的刺激持续时间和2毫秒的延迟。在测量 ITF 之前，在参数测试期间，必须使用两次强度为 2 V 的单抽搐肌肉收缩来确定最佳肌肉张力。这些刺激会导致爪子的多螺旋，并在 VI （图 5） 中的图形上产生输出信号。理想情况下，这些单抽搐曲线具有代表收缩期的快速垂直?…
Discussion
该协议描述了一个先前验证的方法，以获得准确的最大ITF测量TA肌肉在老鼠模型32。实验性神经重建治疗后最大力量的恢复是临床环境的主要兴趣，因为它证明神经不仅再生，而且与目标肌肉建立工作联系。ITF可用于小神经间隙模型，如大鼠坐骨神经模型32，并且通过对协议的一些修改，它也可以用于更大的神经间隙兔子模型33。
<p class="jove_…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
本出版物中报告的研究得到了国家卫生研究院国家神经系统疾病和中风研究所的支持，该研究所获得第1号RO1 NS 102360奖。内容完全由作者负责，不一定代表国家卫生研究院的官方观点。
Materials
| 0.9% Sodium Chloride | Baxter Healthcare Corporation, Deerfield, IL, USA | G130203 | |
| 1 mm Kirshner wires | Pfizer Howmedica, Rutherford, NJ | N/A | |
| Adson Tissue Forceps | ASSI, Westbury, NY, USA | MTK-6801226 | |
| Bipolar electrode cables | Grass Instrument, Quincy, MA | N/A | |
| Bipolar stimulator device | Grass SD9, Grass Instrument, Quincy, MA | N/A | |
| Cotton-tip Applicators | Cardinal Health, Waukegan, IL, USA | C15055-006 | |
| Curved Mosquito forceps | ASSI, Westbury, NY, USA | MTK-1201112 | |
| Force Transducer MDB-2.5 | Transducer Techniques, Temecula, CA | N/A | |
| Gauze Sponges 4×4 | Covidien, Mansfield, MA, USA | 2733 | |
| Ground cable | Grass Instrument, Quincy, MA | N/A | |
| Isoflurane chamber | N/A | N/A | Custom-made |
| Ketamine | Ketalar, Par Pharmaceutical, Chestnut, NJ | 42023-115-10 | |
| LabView Software | National Instruments, Austin, TX | ||
| Loop | N/A | N/A | Custom-made |
| Microsurgical curved forceps | ASSI, Westbury, NY, USA | JFA-5B | |
| Microsurgical scissors | ASSI, Westbury, NY, USA | SAS-15R-8-18 | |
| Microsurgical straight forceps | ASSI, Westbury, NY, USA | JF-3 | |
| Retractor | ASSI, Westbury, NY, USA | AG-124426 | |
| Scalpel Blade No. 15 | Bard-Parker, Aspen Surgical, Caledonia, MI, USA | 371115 | |
| Slim Body Skin Stapler | Covidien, Mansfield, MA, USA | 8886803512 | |
| Subminiature electrode | Harvard Apparatus, Holliston, MA | N/A | |
| Surgical Nerve Stimulator | Checkpoint Surgical LCC, Cleveland, OH, USA | 9094 | |
| Terrell Isoflurane | Piramal Critical Care Inc., Bethlehem, PA, USA | H961J19A | |
| Testing platform | N/A | N/A | Custom-made |
| Tetontomy Scissors | ASSI, Westbury, NY, USA | ASIM-187 | |
| Traceable Big-Digit Timer/Stopwatch | Fisher Scientific, Waltham, MA, USA | S407992 | |
| USB-6009 multifunctional I/O data acquisition (DAQ) device | National Instruments, Austin, TX | 779026-01 | |
| Vacuum Base Holder | Noga Engineering & Technology Ltd., Shlomi, Isreal | N/A | Attached clamp is custom-made |
| Weight (10 g) | Denver Instruments, Denver, CO, USA | 820010.4 | |
| Weight (20 g) | Denver Instruments, Denver, CO, USA | 820020.4 | |
| Weight (50 g) | Denver Instruments, Denver, CO, USA | 820050.4 | |
| Xylazine | Xylamed, Bimeda MTC Animal Health, Cambridge, Canada | 1XYL002 |
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Citazione di questo articolo
Bedar, M., Saffari, T. M., Friedrich, P. F., Giusti, G., Bishop, A. T., Shin, A. Y. Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat. J. Vis. Exp. (172), e61926, doi:10.3791/61926 (2021).