Summary

研究大鼠固定性诱导膝关节弯曲收缩的微型侵入性内固定技术

Published: May 20, 2019
doi:

Summary

在这里,我们提出了一个协议来描述在大鼠模型中膝关节固定的微创技术。这种可重复的规程,基于肌肉间隙分离方式和微型切口技术,适用于研究获得性关节收缩的基本分子机制。

Abstract

由于长期关节固定,联合收缩是骨科常见的并发症。目前,利用内部固定来限制膝关节运动是一种被广泛接受的产生实验收缩的模式。然而,植入应用将不可避免地对动物造成手术创伤。为了开发一种侵入性较低的方法,我们将肌肉间隙分离方法与先前报道的外科手术过程中的迷你切口技巧相结合:在侧大腿和腿部进行两次迷你皮肤切口,然后进行肌肉间隙分离以暴露骨骼表面。大鼠膝关节通过预构造的内部固定在约135°膝盖弯曲时逐渐固定,而不会干扰必要的神经或血管。不出所料,这种简单的技术允许在动物体内快速进行术后康复。X 射线或微 CT 扫描分析证实了内部固定的正确位置。与在反向膝关节中观察到的运动范围相比,固定膝关节的运动范围受到明显限制,这表明该模型的有效性。此外,组织学分析还揭示了后上级膝关节胶囊纤维沉积和粘连的发展。因此,这种微型侵入性模型可能适合模仿固定膝关节收缩的发展。

Introduction

联合合同被定义为在二甲苯关节1,2的被动运动范围(ROM)的限制。目前旨在预防和治疗关节收缩的疗法取得了些成功3,4。然而,获得性联合收缩的基本分子机制在很大程度上仍不为人所知。不同社会群体联合收缩的病因差异很大,包括遗传因素、创伤后状态、慢性病和长期不动6。人们普遍认为,不动是发展获得性联合合同一个关键问题。患有重大联合合同的人最终可能导致身体残疾8。因此,研究获得性联合收缩的潜在病理生理学机制,需要一个稳定、可重复的动物模型。

目前构建的固定诱导膝关节收缩模型主要通过使用非侵入性石膏铸造、外部固定和内部固定来实现。渡边等人9日报告说,在大鼠膝关节上使用石膏石膏固定的可能性。通过穿一件特殊的夹克,大鼠下肢关节的一侧被石膏固定。大鼠膝关节可以保持完全弯曲,没有任何手术创伤10,11。然而,髋关节和踝关节运动也受到这种形式的固定的影响,这可能增加四头肌女性肠12的肌肉萎缩程度。此外,必须通过在设定的时间点更换铸件来避免后肢水肿和充血,这可能会影响不动的连续性。另一种公认的建立膝关节收缩模型的方法是使用外部手术固定。Nagai等人将基什内尔线和钢丝组合成一个外部固定器,将膝关节固定到大约140°的弯曲13。在此方法中,树脂用于覆盖表面以防止皮肤划痕。虽然外部固定固定是健壮和可靠的14,15,皮皮基什内尔线针轨道可能会增加感染的风险16。根据我们自己的经验,使用外部固定技术可能会减少大鼠的日常活动,由于条件舔行为的增加。

另外,Trudel等人描述了一种基于手术内固定17(这种方法从埃文斯和同事18使用的一种方法修改)的在大鼠膝关节中公认的联合收缩模型。值得注意的是,这种方法突出了利用小切口技术尽量减少手术伤口的重要性。联合合同的有效开发已在该模型19中得到证明。然而,关于如何进行最小解剖以暴露骨骼表面的协议仍不清楚20。此外,螺钉钻孔的精确位置尚未完全了解。通过皮下或下肌肉方式植入内部固定仍然是有争议的21。为了解决这些问题,我们修改了这种方法,包括适当的肌肉间隙分离MOD,它允许骨表面的微侵入性暴露和通过肌肉下通道的植入位置。该协议导致大鼠术后快速康复。动物在关节固定后形成有限的关节运动范围,这与组织学分析获得的帽状粘附的形态变化一致。我们还描述了通过 X 射线分析或微 CT 分析确认的钻孔螺钉的确切可能位置。因此,本研究旨在详细描述膝关节收缩模型中的微创技术,该模型由肌肉间隙分离方法与微型切口方法相结合。我们认为,微创技术既能减少动物创伤,又能有效模拟关节弯曲收缩的病理过程。

Protocol

所有程序均按照《实验室动物护理和使用指南》进行,并经中山大学第三附属医院动物护理和使用委员会批准(许可号:02-165-01)。所有的动物实验都是按照”到达”指南进行的。 1. 术前准备 注:图1显示了外科手术的设计。 用塑料板和两个金属螺钉在大约 135° 弯曲处硬固定膝盖接头。注:在不违反关节成分的情况下,在近股骨和远?…

Representative Results

我们观察到,接受微创手术的老鼠术后一天就可以恢复正常饮食。特别是,手术切口有疤痕,没有渗出物 (图5a)。与反向侧相比,手术后两天脚踝和元卡波巴兰关节的肿胀几乎完全消失(图5b)。在大鼠身上没有发现早期感染的迹象。大鼠可以站立和锻炼(图5d)。手术伤口在术后的第十二天完?…

Discussion

这项研究旨在阐明一种分步膝关节固定方法,使用一种微型侵入性技术,允许在手术后动物进行术后快速康复。按照惯例,肌肉间隙分离方法被认为是骨科手术中的微创技术。不出所料,我们发现大鼠在术后一天内就能恢复正常的饮食和活动,这与先前的研究是一致的。此外,手术后没有动脉或神经损伤,证据表明肌肉间隙分离方式确保一个充分和安全的骨暴露方法。虽然使用石膏石膏可以减少侵入性手术…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家自然科学基金(第81772368号)、广东省自然科学基金(第2017A030313496号)和广东省科技计划项目(第2016A020215225号)的资助。No. 2017B090912007)。作者感谢中山大学第八附属医院骨科博士张飞博士在改造过程中的技术援助。

Materials

Anerdian Shanghai Likang Ltd. 310173 antibacterial
Buprenorphine  Shanghai Shyndec Pharmaceutical Ltd. / analgesia 
Carprofen MCE HY-B1227 analgesia 
Cross screwdriver STANLEY PH0*125mm tighten the screws
Electric drill WEGO 185 drill hole(with stainless steel drill 0.9mm;1.0mm)
Microsurgical instruments RWD / Orthopaedic surgical instruments for animals
Neomycin Sigma N6386 antibacterial
Sodium pentobarbital Sigma P3761  anaesthetize
Stainless Steel screws WEGO m1.4*8; m1.2*6 screw(part of internal fixation) 
Syringe  WEGO 3151474 use for plastic plate(part of internal fixation) 
μ-CT  ALOKA Latheta LCT-200 in vivo CT scan

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Jiang, S., Yi, X., Luo, Y., Yu, D., Liu, Y., Zhang, F., Zhu, L., Wang, K. A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats. J. Vis. Exp. (147), e59260, doi:10.3791/59260 (2019).

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