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。

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
<ol><li>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（包括眼科烧灼器、显微外科镊子和显微外科剪刀）高压灭菌，在 102.9 kPa （1.05 kg/cm2） 和 121 °C 下高压灭菌 20 分钟。</li>
	<li>收集所需的手术用品：手术单、乳胶手套、无菌棉、无菌床单、无菌金属箔、作为手术平台的泡沫板、橡皮筋（可以通过撕开医用乳胶手套获得）和胶带（图 1C）（见 材料表）。 注意：为每只小鼠使用一套单独的耗材，包括用于手术区域的注射器和无菌片。</li>
	<li>使用酒精湿巾清洁手术区域和器械（光源、泡沫板和温度维持装置，见 材料表）。用无菌金属箔盖住手术过程中可能需要的器械的旋钮和把手。</li>
	<li>无菌地打开各个器械，并小心地将它们放置在手术区域。激活发芽剂（参见 材料表）和在手术过程中使用的灯。将眼科烧灼器放入发芽器中，加热至250°C20分钟。</li>
</ol>2. 外科手术
<ol><li>按照以下步骤固定鼠标并打开口腔。<ol><li>选择一只体重20-25克、年龄为8-12周龄的雌性C57BL/6J小鼠。在进行任何程序之前将小鼠放置 7 天。</li>
		<li>通过腹膜内注射Zoletil50（80mg / kg）和甲苯噻嗪（5mg / kg）麻醉小鼠（参见 材料表）。将眼科眼药膏涂抹在小鼠的眼睛上。等到没有脚趾捏反应。 注意： 当鼠标无法独立翻转时，鼠标已准备好进行该过程。</li>
		<li>将鼠标固定在衬有无菌床单的泡沫板上。使用胶带将鼠标以仰卧姿势绑在手术平台上（图2A）。</li>
		<li>打开小鼠的口腔。将两根针（26 g x 0.63 英寸）放在眶耳平面的前面，再放置两根针。将橡皮筋放在针头上，然后交叉门牙以保持嘴巴张开。使用显微外科镊子撑开嘴角（图2B）。 注意：确保硬腭清晰暴露。将舌头固定在橡皮筋下方，以防止在随后的实验中阻碍视野和燃烧。</li>
	</ol></li>
	<li>在硬腭上创建口鼻瘘（ONF）（图3A-F）。<ol><li>取回眼科烧灼器，将其加热至250°C20分钟。将烧灼尖端放置在距离上颚中线和第一前磨牙线交汇处 1 毫米处，在中线的硬腭上造成全层粘膜损伤。 注意： 避免烫伤鼠标的舌头。</li>
		<li>几秒钟后，当烧灼尖端周围的粘膜变白时，取下眼科烧灼器。</li>
		<li>将眼科烧灼器置于萌发器中，继续加热至250°C10分钟。重复上一步，扩大边缘周围的伤口，直到其长度达到 2.0 毫米，宽度达到 1.5 毫米。 注意：每次延伸都应遵循最后一次受伤的边缘。使用游标卡尺测量受伤的长度和宽度。损伤应覆盖上颚的 10%。</li>
		<li>使用显微外科剪刀去除伤口周围多余的变性软组织。使用无菌棉止血，防止小鼠吸入性窒息。测量伤口，确保其在中线形成 2.0 x 1.5 毫米的全层硬腭粘膜损伤。</li>
	</ol></li>
</ol>3. 术后护理
<ol><li>在术后觉醒时给予小鼠美洛昔康，剂量为5mg / kg / d，持续3天，皮下注射29。</li>
	<li>将鼠标放在温度维持装置上，直到它完全恢复意识。 注意： 确保鼠标的位置有利于呼吸。每10-15分钟旋转一次小鼠，以防止血液淤积或肺叶塌陷。一旦老鼠热身，将其放回笼子里。在笼子底部提供无菌果冻和辐照饲料供小鼠食用。</li>
</ol>4. 口鼻瘘的形成验证
注意：口鼻瘘 （ONF） 创建的成功率在手术后的第 7 天进行评估。
<ol><li>准备必要的手术用品：橡皮筋、胶带、注射器、手术单、乳胶手套、无菌床单、无菌金属箔和泡沫板。</li>
	<li>佩戴手术单和无菌手套以保持无菌状态。用酒精对泡沫板、光源、保温装置进行消毒。</li>
	<li>通过腹膜内注射Zoletil50（80mg / kg）诱导全身麻醉。等到没有脚趾捏反应。使用步骤 2.1.3 和 2.1.4 中描述的相同方法固定鼠标并暴露硬腭。</li>
	<li>通过确保隔膜在伤口部位仍然可见来进行解剖结构验证，表明ONF创建成功（图4A，B）。</li>
	<li>进行功能验证：关闭小鼠的口腔，并使用无菌注射器将无菌水注入其口腔。当液体从小鼠的鼻孔流出时，ONF的成功产生得到确认。</li>
	<li>将鼠标放在温度维持装置（37°C）上，直到它完全恢复意识。每10-15分钟旋转一次小鼠，以防止血液淤积或肺叶塌陷。</li>
</ol>

开发用于研究口鼻瘘的有效小鼠模型

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作者没有什么可透露的。

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，?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
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			<td>Electron Microscopy Sciences&#160;</td>
			<td>66118-20</td>
			<td>Heating and disinfection equipment</td>
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			<td>Latex gloves</td>
			<td>Allmed</td>
			<td></td>
			<td>or similar</td>
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			<td>Lights</td>
			<td>Olympus</td>
			<td>A1813</td>
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			<td>Meloxicam</td>
			<td>MedChemExpress</td>
			<td>HY-B0261</td>
			<td>crushed; 5 mg/kg</td>
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			<td>Microsurgical instruments (scissors and tweezers)</td>
			<td>Jiangsu Tonghui Medical Devices Co.</td>
			<td>M-Y-0087</td>
			<td>Surgical instrument</td>
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			<td>Ophthalmologic cautery</td>
			<td>Suqian Wenchong Medical Equipment Co.</td>
			<td>1.00234E+13</td>
			<td>Surgical instrument</td>
		</tr>
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			<td>Sterile cotton,</td>
			<td>Yancheng Begu Technology Co.</td>
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			<td>or similar</td>
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</table>

establishment of an oronasal fistula mice model

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。

Oronasal fistula (ONF), an abnormal opening between the oral and nasal cavities, clinically manifests as a defect in a structural area from the alveolar process to the uvula, which commonly occurs as a complication following cleft palate repair1. Patients with ONF experience food reflux, articulation disorders, and impaired velopharyngeal function, significantly impacting their quality of life2,3,4. The rate of post-operative ONF ranges from 2.4% to 55% due to factors such as cleft width, Veau type, and surgical method5,6,7,8. Additionally, the recurrence rate after ONF repair is high, ranging from 0% to 43%9.
Several novel treatments have recently shown promise in the field of ONF, including different materials, drugs, and novel techniques10,11,12,13,14,15,16,17. Accurate evaluation of therapeutic effects is essential as it provides the basis for selecting and further developing ONF treatments. However, obtaining a valid assessment in the short term for therapeutic effects other than surgery is challenging, as the characteristics of ONFs vary among different patients. Therefore, establishing an ONF disease model is necessary to verify the effectiveness of these treatment methods.
For several decades, researchers have generated the oronasal fistula (ONF) model in various animal species, including rats18,19, piglets20,21, minipigs22, and dogs23, as these species possess a substantial hard palate suitable for surgical manipulation. However, mice have a genetic sequence and whole genome similar to that of humans, making them an important model for researching and developing new drugs24,25,26. Furthermore, mice offer little variation from batch to batch, making them a favorable choice for establishing the ONF model12,13,27.
However, the detailed steps for creating ONF were not described, and the stability of the ONF size was not taken into consideration. Additionally, the verification of ONF formation relied solely on observation28, without ensuring direct communication between the oral and nasal cavities. It was not demonstrated through other means, such as the mouse&#39;s loss of body weight due to difficulties in eating caused by the ONF. Furthermore, normal variation in wound size was not considered, which is crucial for studies on drugs or materials that promote or inhibit wound healing. Therefore, there is a strong need to establish a stable and validated ONF model.
The objective of this study was to develop a practical ONF model that addresses the aforementioned issues, with the hope that this protocol will serve as the foundation for future research on the mechanisms of palatal wound healing and novel treatments for ONF.

作者聚焦：研究小鼠口鼻瘘模型的伤口愈合 

口鼻瘘小鼠模型的建立

Our research has focused on how to establish the stable and standardized mouse model of oronasal fistula. This protocol has also demonstrated the body weight changes, wound size changes, and the histologic features of the oral nasal fistula formation. The current experimental challenges of establishing oronasal fistula models are the mice mortality by suffocation or blood loss as well as the standardization of the wound size.Our protocol significantly reduces the amount of bleeding in mice during surgery relative to the traditional use of a biopsy punch. This reduces the risk of suffocation due to severe bleeding. Additionally, our experimental method is also simpler and less technically sensitive.Our future research aims to focus on observing the inflammatory response during oronasal fistula formation. We also plan on developing novel strategies to reduce the inflammatory response while promoting palatal wound healing to reduce the incidence of oronasal fistula.

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。...

Watch this Scientific Journal Video about Investigating Wound Healing in Mice Models of Oronasal Fistulas at JoVE.com

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。

This study presents a method utilizing heated ophthalmologic cautery to develop a viable model for investigating oronasal fistulas. C57BL/6 mice were used ...

我们的研究重点是如何建立稳定和标准化的口鼻瘘小鼠模型。该方案还证明了体重变化、伤口大小变化和口腔鼻瘘形成的组织学特征。目前建立口鼻瘘模型的实验挑战是小鼠因窒息或失血而死亡以及伤口大小的标准化。与传统的活检打孔相比，我们的方案显着减少了手术期间小鼠的出血量。这降低了因严重出血而窒息的风险。此外，我们的实验方法也更简单，技术敏感性更低。我们未来的研究旨在专注于观察口鼻瘘形成过程中的炎症反应。我们还计划开发新的策略来减少炎症反应，同时促进腭伤口愈合，以降低口鼻瘘的发生率。

establishment-of-an-oronasal-fistula-mice-model

Research

JoVE Journal

Medicine

Establishment of an Oronasal Fistula Mice Model

814 次观看.  Sichuan University. 我们的研究重点是如何建立稳定和标准化的口鼻瘘小鼠模型。该方案还证明了体重变化、伤口大小变化和口腔鼻瘘形成的组织学特征。目前建立口鼻瘘模型的实验挑战是小鼠因窒息或失血而死亡以及伤口大小的标准化。与传统的活检打孔相比，我们的方案显着减少了手术期间小鼠的出血量。这降低了因严重出血而窒息的风险。此外，我们的实验方法也更简单，技术敏感?...

视频: 作者聚焦：研究小鼠口鼻瘘模型的伤口愈合 

This study presents a method utilizing heated ophthalmologic cautery to develop a viable model for investigating oronasal fistulas. C57BL/6 mice were used to establish the oronasal fistula (ONF) model. To create the ONF, the mice were anesthetized, immobilized, and their hard palates were exposed. During the surgical procedure, a 2.0 x 1.5 mm full-thickness mucosal injury was induced in the midline of the hard palate using ophthalmologic cautery. It was crucial to control the size of the ONF and minimize bleeding in order to ensure the success of the experiment. Verification of the ONF model's effectiveness was conducted on the 7th-day post-operation, encompassing both anatomical and functional assessments. The presence of the nasal septum within the oral cavity and the outflow of sterile water from the nostrils upon injection into the oral cavity confirmed the successful establishment of the ONF model. The model demonstrated a practical and successful oronasal fistula, characterized by a low mortality rate, significant weight changes, and minimal variation in ONF size. Future studies may consider adopting this methodology to elucidate the mechanisms of palate wound healing and explore novel treatments for oronasal fistulas.

This article outlines a step-by-step procedure for establishing a mice model with an oronasal fistula. The oronasal fistula was created by employing heated ophthalmologic cautery to damage the midline portion of the hard palate, resulting in the formation of an opening between the oral and nasal cavities.

科研

医学

Development of a Mouse Model for Studying Oronasal Fistula Using Opthalmologic Cautery

Begin by securing an anesthetized female mouse to a foam board covered with sterile sheets. Then, carefully tape the mouse in a supine position to a surgical platform. Position two needles in front of the orbital airplane.Place two more needles behind it. Next, place a rubber band around the needles and cross the incisors to hold the mouth open. Use microsurgical tweezers to prop open the corners of the mouth.Proceed to retrieve the heated ophthalmologic cautery. Now position the cautery tip one millimeter from the midline intersection of the palate and first premolar to create a full thickness mucosal injury to the hard palate. Remove the cautery when the mucosa around the tip turns white.Heat the ophthalmologic cautery to 250 degrees Celsius in the germinator for 10 minutes. Then, place it on the hard palate and enlarge the wound around the edges. Finally, use microsurgical scissors to excise any excess denatured soft tissue surrounding the wound.Afterward, apply sterile cotton to stop bleeding and prevent inhalation asphyxiation in the mouse. Notable variability was observed in the size of the generated oral nasal fistula. The wound size showed a significant variation on the seventh day post-surgery.A significant reduction in the body weights of the mice was also observed on day seven, which is approximately 25%Histological analysis of the oral nasal fistula revealed a loss of hard palate mucosa, denuded bone, and oronasal fistula formation.