口鼻瘘小鼠模型的建立
1State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases and Department of Oral Maxillofacial Surgery, West China Hospital of Stomatology,Sichuan University, 2State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases and Department of Head and Neck Oncology, West China Hospital of Stomatology,Sichuan University, 3State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases and Eastern Clinic, West China Hospital of Stomatology,Sichuan University, 4State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases and Department of Oral Prosthodontics, West China Hospital of Stomatology,Sichuan University
Summary
本文概述了建立具有口鼻瘘的小鼠模型的分步程序。口鼻瘘是通过采用加热的眼科烧灼术破坏硬腭的中线部分而形成的，导致口腔和鼻腔之间形成开口。
Abstract
本研究提出了一种利用加热眼科烧灼术开发研究口鼻瘘的可行模型的方法。采用C57BL/6小鼠建立口鼻瘘（ONF）模型。为了产生ONF，小鼠被麻醉，固定，并暴露它们的硬腭。在手术过程中，使用眼科烧灼术在硬腭中线诱导 2.0 x 1.5 mm 的全层粘膜损伤。控制ONF的大小并尽量减少出血以确保实验成功至关重要。在术后第 7 天对 ONF 模型的有效性进行了验证，包括解剖学和功能评估。口腔内鼻中隔的存在以及注射到口腔时从鼻孔流出的无菌水证实了ONF模型的成功建立。该模型展示了实用且成功的口鼻瘘，其特点是死亡率低、体重变化显着且 ONF 大小变化最小。未来的研究可能会考虑采用这种方法来阐明腭伤口愈合的机制，并探索口鼻瘘的新疗法。
Introduction
口鼻瘘 （ONF） 是口腔和鼻腔之间的异常开口，临床表现为从肺泡突到悬雍垂的结构区域缺陷，通常作为腭裂修复后的并发症发生1.ONF 患者会出现食物反流、发音障碍和腭咽功能受损，显着影响他们的生活质量 2,3,4。术后 ONF 的发生率在 2.4% 至 55% 之间，这是由于裂隙宽度、Veau 类型和手术方法5、6、7、8 等因素造成的。此外，ONF修复后的复发率很高，从0%到43%不等9。
最近，几种新的治疗方法在ONF领域显示出前景，包括不同的材料、药物和新技术10,11,12,13,14,15,16,17。准确评估治疗效果至关重要，因为它为选择和进一步开发ONF治疗提供了基础。然而，在短期内获得手术以外的治疗效果的有效评估具有挑战性，因为不同患者的 ONF 特征各不相同。因此，建立ONF疾病模型对于验证这些治疗方法的有效性是必要的。
几十年来，研究人员在各种动物物种中生成了口鼻瘘 （ONF） 模型，包括大鼠18,19、仔猪20、21、迷你猪 22 和狗 23，因为这些物种具有适合手术操作的大量硬腭。然而，小鼠的基因序列和全基因组与人类相似，使其成为研究和开发新药的重要模型24,25,26。此外，小鼠在批次之间几乎没有变化，使它们成为建立ONF模型12,13,27的有利选择。
但是，没有描述创建ONF的详细步骤，也没有考虑ONF大小的稳定性。此外，ONF形成的验证仅依赖于观察28，而没有确保口腔和鼻腔之间的直接沟通。它没有通过其他方式得到证明，例如由于ONF引起的进食困难导致小鼠体重减轻。此外，没有考虑伤口大小的正常变化，这对于研究促进或抑制伤口愈合的药物或材料至关重要。因此，迫切需要建立一个稳定且经过验证的ONF模型。
本研究的目的是开发一个实用的 ONF 模型来解决上述问题，希望该协议将成为未来研究腭伤口愈合机制和 ONF 新疗法的基础。
Protocol
本研究中的所有动物手术均由四川大学华西口腔医学院伦理委员会审查批准。本研究采用成年C57BL/6小鼠（雌性）。 1. 手术准备 收集手术所需的手术器械：生发器，眼科烧灼器，显微外科剪刀，显微外科镊子，注射器和针头（26g×0.63英寸）（图1A，B）（见材料表）。注意：在外科手术之前，将手术器械（?…
Representative Results
为了评估该实验方法的可行性和稳定性，对 10 只小鼠进行了相同的程序，并观察了死亡率、伤口大小、体重和组织学分析的变化。小鼠在第7天被安乐死。 该手术的死亡率较低。眼科烧灼器和萌发器，如图1A-C所示，是本实验中使用的关键仪器。ONF模型是根据提供的协议创建的。在手术的10只小鼠中，只有1只在手术后第7天死亡。…
Discussion
研究人员探索了治疗 ONF10、11、12、13、14、15、16、17 的各种材料、药物和新技术。随着外科手术的进步，ONF的发病率和复发率已经降低。然而，由于该疾病的独特性，临床上ONF患者的数量有限，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了四川大学华西口腔医院研究与发展计划（RD-02-202107）、四川省科技支持计划（2022NSFSC0743）和四川省博士后科学基金（TB2022005）资助。
Materials
| Germinator | Electron Microscopy Sciences | 66118-20 | Heating and disinfection equipment |
| Latex gloves | Allmed | or similar | |
| Lights | Olympus | A1813 | |
| Meloxicam | MedChemExpress | HY-B0261 | crushed; 5 mg/kg |
| Microsurgical instruments (scissors and tweezers) | Jiangsu Tonghui Medical Devices Co. | M-Y-0087 | Surgical instrument |
| Ophthalmologic cautery | Suqian Wenchong Medical Equipment Co. | 1.00234E+13 | Surgical instrument |
| Sterile cotton, | Yancheng Begu Technology Co. | or similar | |
| Sterile metal foil | Biosharp | or similar | |
| Sterile sheets | 3M | XH003801129 | or similar |
| Surgical drapes | Yancheng Begu Technology Co. | or similar | |
| Syringes | Yancheng Begu Technology Co. | S-015301 | or similar |
| Tape | Bkmamlab | or similar | |
| Temperature maintenance device | Harvard Apparatus | LE-13-2104 | |
| Zoletil50 | Virbac | 80 mg/kg |
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Cite This Article
Chen, J., Yin, J., Zhang, S., Zhuang, S., Yang, R., Xu, Y., Zheng, Q., Shi, B., Huang, H. Establishment of an Oronasal Fistula Mice Model. J. Vis. Exp. (199), e65578, doi:10.3791/65578 (2023).