This work was supported by the Research and Development Program, West China Hospital of Stomatology, Sichuan University (RD-02-202107), Sichuan Province Science and Technology Support Program (2022NSFSC0743), and Sichuan Postdoctoral Science Foundation (TB2022005) grant to H. Huang.

All animal procedures in this study were reviewed and approved by the Ethical Committee of the West China School of Stomatology, Sichuan University. Adult C57BL/6 mice (female) were used for the present study.
1. Surgical preparation
<ol>
	<li>Gather the necessary surgical instruments for the procedure: germinator, ophthalmologic cautery, microsurgical scissors, microsurgical tweezers, syringes, and needles (26 g x&#160; 0.63 inch) (Figure 1A<st...

Development of an Effective Mice Model for Studying Oronasal Fistulas

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Postoperative+Outcomes+in+Two+Cleft+Palate+Repair+Techniques+without+Relaxing+Incisions.">Evaluation of Postoperative Outcomes in Two Cleft Palate Repair Techniques without Relaxing Incisions.</a> Plastic and Reconstructive Surgery. , (2023).</li><li>Sakran, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+late+cleft+palate+repair+by+a+modified+technique+without+relaxing+incisions.">Evaluation of late cleft palate repair by a modified technique without relaxing incisions.</a> Journal of Stomatology, Oral and Maxillofacial Surgery. 124 (4), 101403 (2023).</li><li>Sakran, K. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+and+soft+tissue+wound+healing:+a+systematic+review.">Platelet-rich fibrin and soft tissue wound healing: a systematic review.</a> Tissue Engineering Part B: Reviews. 23 (1), 83-99 (2017).</li><li>Ballestas, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+hard+palate+wound+healing+using+immune+modulatory+autotherapies.">Improving hard palate wound healing using immune modulatory autotherapies.</a> Acta Biomaterialia. 91, 209-219 (2019).</li><li>Ferreira, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+enhances+early+palatal+wound+healing+in+mice.">Electrical stimulation enhances early palatal wound healing in mice.</a> Archives of Oral Biology. 122, 105028 (2021).</li><li>Lindley, L. E., Stojadinovic, O., Pastar, I., Tomic-Canic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+Biomarkers+for+Wound+Healing.">Biology and Biomarkers for Wound Healing.</a> Plastic and Reconstructive Surgery. 138 (3 Suppl), 18s-28s (2016).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Additive+Manufacturing+of+a+Superlight+Obturator+for+Large+Oronasal+Fistula+in+Pediatric+Patient.">Rapid Additive Manufacturing of a Superlight Obturator for Large Oronasal Fistula in Pediatric Patient.</a> Laryngoscope. 133 (6), 1507-1512 (2022).</li><li>Leenstra, T. S., Kuijpers-Jagtman, A. M., Maltha, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+healing+process+of+palatal+tissues+after+palatal+surgery+with+and+without+implantation+of+membranes:+an+experimental+study+in+dogs.">The healing process of palatal tissues after palatal surgery with and without implantation of membranes: an experimental study in dogs.</a> Journal of Materials Science: Materials in Medicine. 9 (5), 249-255 (1998).</li><li>In de Braekt, M. M., van Alphen, F. A., Kuijpers-Jagtman, A. M., Maltha, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+wound+contraction+after+palatal+surgery+and+implantation+of+poly-(L-lactic)+acid+membranes+in+beagle+dogs.">Wound healing and wound contraction after palatal surgery and implantation of poly-(L-lactic) acid membranes in beagle dogs.</a> Journal of Oral and Maxillofacial Surgery. 50 (4), 359-365 (1992).</li><li>Suragimath, G., Krishnaprasad, K. R., Moogla, S., Sridhara, S. U., Raju, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+carbonated+drink+on+excisional+palatal+wound+healing:+A+study+on+Wistar+rats.">Effect of carbonated drink on excisional palatal wound healing: A study on Wistar rats.</a> Indian Journal of Dental Research. 21 (3), 330-333 (2010).</li><li>Zhu, T., Park, H. C., Son, K. M., Yang, H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dimethyloxalylglycine+on+wound+healing+of+palatal+mucosa+in+a+rat+model.">Effects of dimethyloxalylglycine on wound healing of palatal mucosa in a rat model.</a> BMC Oral Health. 15 (1), 60 (2015).</li><li>Kirschner, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+oronasal+fistulae+with+acellular+dermal+matrices.">Repair of oronasal fistulae with acellular dermal matrices.</a> Plastic and Reconstructive Surgery. 118 (6), 1431-1440 (2006).</li><li>Rohleder, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+oronasal+fistulae+by+interposition+of+multilayered+amniotic+membrane+allograft.">Repair of oronasal fistulae by interposition of multilayered amniotic membrane allograft.</a> Plastic and Reconstructive Surgery. 132 (1), 172-181 (2013).</li><li>Kesting, M. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+delivery+of+FTY720+induces+neutrophil+activation+through+chemokine+signaling+in+an+oronasal+fistula+model.">Local delivery of FTY720 induces neutrophil activation through chemokine signaling in an oronasal fistula model.</a> Regenerative Engineering and Translational Medicine. 7 (2), 160-174 (2021).</li><li>Antiorio, A. T. F. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+meloxicam+to+improve+the+welfare+of+mice+in+research:+a+systematic+review+(2000+-+2020).">Administration of meloxicam to improve the welfare of mice in research: a systematic review (2000 - 2020).</a> Veterinary Research Communications. 46 (1), 1-8 (2022).</li></ol>

Researchers have explored various materials, drugs, and novel techniques for treating ONF10,11,12,13,14,15,16,17. With advancements in surgical procedures, the incidence and recurrence of ONF have been reduced. However, due to the unique characteristics of ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Germinator</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>66118-20</td>
			<td>Heating and disinfection equipment</td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>Allmed</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Lights</td>
			<td>Olympus</td>
			<td>A1813</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>MedChemExpress</td>
			<td>HY-B0261</td>
			<td>crushed; 5 mg/kg</td>
		</tr>
		<tr>
			<td>Microsurgical instruments (scissors and tweezers)</td>
			<td>Jiangsu Tonghui Medical Devices Co.</td>
			<td>M-Y-0087</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Ophthalmologic cautery</td>
			<td>Suqian Wenchong Medical Equipment Co.</td>
			<td>1.00234E+13</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Sterile cotton,</td>
			<td>Yancheng Begu Technology Co.</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Sterile metal foil</td>
			<td>Biosharp</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Sterile sheets</td>
			<td>3M</td>
			<td>XH003801129</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Surgical drapes</td>
			<td>Yancheng Begu Technology Co.</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Yancheng Begu Technology Co.</td>
			<td>S-015301</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Bkmamlab</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Temperature maintenance device</td>
			<td>Harvard Apparatus&#160;</td>
			<td>LE-13-2104</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil50</td>
			<td>Virbac</td>
			<td></td>
			<td>80 mg/kg</td>
		</tr>
	</tbody>
</table>

establishment of an oronasal fistula mice model

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.

Author Spotlight: Investigating Wound Healing in Mice Models of Oronasal Fistulas 

Establishment of an Oronasal Fistula Mice Model

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.

To assess the feasibility and stability of this experimental method, the same procedure was performed on ten mice, and observations were made regarding mortality, changes in wound size, body weight, and histologic analysis. The mice were euthanized on day 7.
The procedure exhibited a low mortality rate. The ophthalmologic cautery and germinator, depicted in Figure 1A-C, were the key instruments utilized in this experiment. The ONF...

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

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.

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

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