Name: Development of a Mouse Model for Studying Oronasal Fistula Using Opthalmologic Cautery
Uploaded: 2023-09-08T14:25:00
Description: Watch this Scientific Journal Video about Development of a Mouse Model for Studying Oronasal Fistula Using Opthalmologic Cautery at JoVE.com

development of a mouse model for studying oronasal fistula using opthalmologic cautery

Development of an Effective Mice Model for Studying Oronasal Fistulas

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.

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.

Watch this Scientific Journal Video about Development of a Mouse Model for Studying Oronasal Fistula Using Opthalmologic Cautery at JoVE.com

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.

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Research

JoVE Journal

Medicine

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.

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