踝距下复杂关节不稳定的小鼠模型
Summary
踝距下复合关节 （ASCJ） 是足部的核心，在日常活动中起着关键作用。运动损伤通常会导致该关节不稳定。在这里，我们描述了韧带横断诱导的ASCJ不稳定性的小鼠模型。
Abstract
踝关节扭伤可能是日常生活中最常见的运动损伤，通常会导致踝距下复合关节 （ASCJ） 不稳定，从长远来看，最终会导致创伤后骨关节炎 （PTOA）。然而，由于损伤机制的复杂性和临床表现，如瘀斑、血肿或外侧足压痛，对ASCJ不稳定的诊断和治疗尚无临床共识。由于小鼠后足骨骼和韧带的肌肉骨骼结构与人类相当，因此通过ASCJ周围韧带的横断建立了小鼠ASCJ不稳定的动物模型。该模型通过一系列行为测试和组织学分析得到了充分验证，包括平衡木测试、足迹分析（评估小鼠的运动水平和平衡能力）、热伤害感受评估（评估小鼠的足部感觉功能）、显微计算机断层扫描 （CT） 扫描和关节软骨切片染色（评估小鼠关节软骨损伤和退化）。成功建立ASCJ不稳定小鼠模型将为损伤机制的临床研究提供有价值的参考，并为踝关节扭伤提供更好的治疗选择。
Introduction
踝关节扭伤是全球最常见的运动损伤之一。据估计，美国每天有 10,000 人受伤1，其中运动相关伤害占 15%-45%2。在美国，与治疗踝关节扭伤相关的医疗费用每年达 42 亿美元 3,4,5。慢性足部不稳是踝关节扭伤后的常见问题，约占踝关节扭伤的 74% 6，包括踝关节或距下关节不稳。然而，由于临床症状和体征相似，医务人员在临床上很难区分慢性踝关节不稳是否也伴有距下关节不稳，因此容易漏诊慢性距下关节不稳。因此，慢性踝距下复合关节 （ASCJ） 不稳定（一种特定类型的慢性足部不稳定，包括慢性踝关节不稳定和慢性距下不稳定）的真实发生率可能高于报告的 7,8,9。如果不及时治疗，慢性踝距下复合关节不稳定会导致反复踝关节扭伤，导致踝关节扭伤和慢性踝距下复合关节不稳定的恶性循环。长期慢性踝距下复合体不稳定可导致 ASCJ 退化和创伤后骨关节炎，严重时会影响邻近关节10.对于这些疾病，目前的临床治疗主要是保守治疗，此外还有韧带修复和韧带重建等手术治疗方法11,12。
ASCJ 是足部的核心结构，在运动13 期间保持身体平衡。已经对踝关节和距下关节的结构进行了广泛的研究分别 14,15,16,17。然而，对整个踝距下关节的研究很少见。大约四分之一的踝关节损伤病例与距下关节损伤有关18。由于ASCJ不稳定的损伤机制复杂，临床上对诊断和治疗尚无共识。考虑到目前临床踝关节损伤的现状，需要一种更科学的方法，将踝关节和距下关节作为一个整体进行研究，从而为研究足部疾病提供新的认识。
由于小鼠后足在肌肉骨骼水平上的解剖结构与人足的解剖结构相当19，因此在几项研究中，已经实施了用于足/踝研究的小鼠模型10,19。Chang 等 19 成功开发了三种不同的踝骨关节炎小鼠模型。受小鼠模型中踝关节不稳定性成功建立的启发，我们建立了踝距下复合体不稳定性的小鼠模型，假设小鼠后足部分韧带的横断会导致ASCJ的机械不稳定，从而导致ASCJ的创伤后骨关节炎（PTOA）。ASCJ不稳动物模型可用于踝关节不稳和距下不稳的治疗，比目前使用的单纯踝关节不稳模型7、8、9、19更符合临床实际情况。为了验证这一假设，设计了两个韧带横断诱导的ASCJ不稳定的小鼠模型。采用平衡木试验、足迹分析、热伤害感受评估等感觉运动功能结果评价模型的可行性，采用显微计算机断层扫描（CT）和组织学染色评估小鼠关节软骨损伤和变性。ASCJ不稳定性小鼠模型的成功建立，不仅为研究足部疾病提供了新的认识，也为损伤相关机制的临床研究提供了有价值的参考，为踝关节扭伤提供了更好的治疗选择，有助于进一步研究该疾病。
Protocol
所有动物研究均按照《实验动物护理和使用指南》进行，并得到苏州大学机构动物护理和使用委员会的批准。 1. 外科手术 将21只6周龄的C57BL/6雄性小鼠分为三组：颈横韧带和距腓前韧带组，颈横韧带和三角韧带组，假手术组。确保所有小鼠都是在符合特定无病原体（SPF）标准的环境中饲养的。 在实验前2周使小鼠适应新的饲养环境（12小时/ 12小时的…
Representative Results
利用在线统计分析工具对相关数据进行统计分析。采用单因素方差分析，将满足正态分布和方差均匀性两个检验的数据用于进一步的统计分析。如果数据不符合两个检验，则采用Kruskal-Wallis检验进行统计分析。数据表示为平均值±标准差 （SD）， p < 0.05 被认为具有统计学意义。 平衡木试验对每组小鼠在每阶段通过平衡木两次的平均所需时间进行统计分析…
Discussion
本研究通过横断CL+ATFL或CL+DL成功构建了2个ASCJ不稳定性小鼠模型。小鼠通过平衡木的时间在手术后8周和12周显着增加，这与Hubbard-Turner团队通过切割踝关节外侧韧带获得的结果相似23,24。在右足滑动试验中，我们观察到两组断韧带小鼠的滑行次数明显高于假韧带组小鼠，并且在手术后12周滑动次数达到最大值，从而提示两组断韧带小鼠可能患有ASCJ不稳定?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究得到了江苏省政府奖学金项目和江苏省高等学校学术项目发展项目（PAPD）的支持。
Materials
| 5-0 Surgical Nylon Suture | Ningbo Medical Needle Co., Ltd. | 191104 | |
| Acidic ethanol differentiation solution (1%) | Shanghai Yuanye Biotechnology Co., Ltd. | R20778 | |
| Adhesive slides | Jiangsu Shitai Company | ||
| Ammonia solution (1%) | Shanghai Yuanye Biotechnology Co., Ltd. | R20788 | |
| Anhydrous ethanol | Shanghai Sinopharm Group Chemical Reagent Co., Ltd. | ||
| Aqueous acetic acid (1%) | Shanghai Yuanye Biotechnology Co., Ltd. | R20773 | |
| Black cube cassette | Shanghai Yizhe Instrument Co., Ltd. | ||
| Centrifuge tube 15ml | Beijing Soleibo Technology Co., Ltd. | YA0476 | |
| Centrifuge tube 50ml | Beijing Soleibo Technology Co., Ltd. | YA0472 | |
| Cover glass | Jiangsu Shitai Company | ||
| CTAn software | Blue scientific | micro-CT analysis software | |
| Dataview software | AEMC instruments | commercial data analysing software | |
| Disodium ethylenediaminetetraacetate (EDTA-2Na) | Beijing Soleibo Technology Co., Ltd. | E8490 | |
| Electric incubator | Suzhou Huamei Equipment Factory | ||
| Embedding paraffin | Leica, Germany | 39001006 | |
| Eosin staining solution (alcohol soluble, 1%) | Shanghai Yuanye Biotechnology Co., Ltd. | R30117 | |
| Fast green staining solution | Sigma-Aldrich, USA | F7275 | |
| Gait paper | Baoding Huarong Paper Factory | ||
| GraphPad Prism 8.0 | Graphpad software | online statistical analysis tools | |
| Iodophor cotton balls | Qingdao Hainuo Bioengineering Co., Ltd. | ||
| Leica 818 blade | Leica, Germany | ||
| Micro-CT | Skyscan, Belgium | SkyScan 1176 | |
| Micromanipulation microscope | Suzhou Omet Optoelectronics Co., Ltd. | ||
| Mimics software | Materialise | 3D medical image processing software | |
| Modified Harris Hematoxylin Stain | Shanghai Yuanye Biotechnology Co., Ltd. | R20566 | |
| Mouse anti-mouse type II collagen | American Abcam Company | ||
| NaOH | Shanghai Sinopharm Group Chemical Reagent Co., Ltd. | ||
| N-butanol | Shanghai Sinopharm Group Chemical Reagent Co., Ltd. | ||
| Neutral formalin fixative (10%) | Shanghai Yuanye Biotechnology Co., Ltd. | ||
| Neutral resin | Sigma-Aldrich, USA | ||
| Nrecon reconstrcution software | Micro Photonics Inc. | ||
| Oaks hair clipper | Oaks Group Co., Ltd. | ||
| Paraffin Embedding Machine | Leica, Germany | ||
| PH meter | Shanghai Leitz Company | ||
| Phosphate Buffered Saline (PBS) | American Biosharp | ||
| Physiological saline (for mammals, sterile) | Shanghai Yuanye Biotechnology Co., Ltd. | R22172 | |
| Safranin O-staining solution | Sigma-Aldrich, USA | HT90432 | |
| Saline (0.9%) | Shanghai Baxter Medical Drug Co., Ltd. | 309107 | |
| Shaker | Haimen Qilin Bell Instrument Manufacturing Co., Ltd. | 2008779 | |
| SPSS 23 | IBM | online statistical analysis tools | |
| Tablet machine | Leica, Germany | ||
| Tissue slicer | Leica, Germany | ||
| Ugo Basile | Ugo Basile Biological Research Company | ||
| Upright fluorescence microscope | Zeiss Axiovert, Germany | ||
| U-shaped plastic channel | Shanghai Yizhe Instrument Co., Ltd. | ||
| Veterinary eye ointment | Pfizer | ||
| Xylene | Shanghai Sinopharm Group Chemical Reagent Co., Ltd. | ||
| YLS-10B Wheel Fatigue Tester | Jinan Yiyan Technology Development Co., Ltd. |
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Citer Cet Article
Wang, S., Liu, P., Hua, C., Zhang, H., Yu, J. A Mouse Model of Ankle-Subtalar Complex Joint Instability. J. Vis. Exp. (188), e64481, doi:10.3791/64481 (2022).