preparation of the mouth gag for a mouse pulpitis model

Erstellung eines Mausmodells für eine Pulpaentzündung unter Verwendung eines neuartigen Mundknebels 

The dental pulp, an integral part of the tooth, is responsible for multiple essential functions such as nutrient supply, dentin formation, sensory function, and defense reactions1. Nevertheless, the dental pulp, surrounded by hard tissue, is susceptible to injuries and damages from deep caries, pulpitis, trauma, or subsequent therapies2,3. The absence of functional dental pulp increases the risk of tooth fragility4. Moreover, the loss of pulp vitality in young permanent teeth can adversely affect tooth maturation, and current denture techniques fail to restore the neural feedback offered by healthy pulp4. This situation has led researchers to explore alternative solutions for managing inflamed pulp beyond mere removal.
In 2007, Murray et al. initiated the application of tissue engineering in regenerative endodontics, thereby sparking increased interest in pulp preservation and regeneration5. However, inflamed pulp tissue poses a challenge as cells release inflammatory factors such as IL-6, which recruit inflammatory cells and results in cell necrosis, loss of pulp vitality, and complications in functional recovery6,7. Understanding inflammation and the associated cell death is, therefore, crucial for advancements in the preservation of vital pulp. There are a number of experiments that have been conducted to explore the molecular biology of the inflame pulp in vivo or in vitro8,9. Though in vitro&#160;experiments like 2D or 3D cell cultures have been developed for years and are becoming mature and widely used to test reactions of pulp cells to inflammatory factors, these experiments cannot reflect the interaction between pulp tissue and the systemic immune system10. If the phenomenon being studied is derived from cells of other tissue origin like immune, vascular and nervous system, then pure pulp cell culture will lead to a dead end. Therefore, in vivo experiments are very necessary and referential.
Mice have increasingly become a common choice in inflammation research in vivo&#160;due to their cost-effectiveness, high fertility, and vitality. However, a comprehensive protocol for mice pulpitis model is currently absent, which can serve as a reference. The small size of mice and their sensitivity to stimulation pose significant challenges during experimental procedures. Observing the minuscule teeth concealed deep within the mouse mouth often necessitates the use of a cantilever microscope, notwithstanding the more common presence of desktop microscopes in laboratories. The absence of a mouth opener requires assistance from others. To address this, the group has devised a mouth-gag using readily available materials which aims to provide a standardized and reproducible protocol for constructing the mice pulpitis model. This article details the procedure, covering preoperative preparation, immobilization, pulp exposure surgery, and sample collection on C57 mice. This protocol recommends the use of the mouth-gag, providing information on its structure, production, and application to facilitate other researchers in replicating the procedure.

Autor im Rampenlicht: Verbesserung der Zahnpulpaforschung mit verbesserten Mausmodellen

Etablierung eines murinen Pulpa-Expositionsmodells mit einem neuartigen Mundknebel für die Pulpitisforschung

Individual experiments like 2D or 3D cell cultures have been widely used to test the reactions of dental pulp cells. These experiments cannot reflect the interaction between pulp tissue and the systemic immune system. Therefore, in vivo experiments are becoming increasingly popular in endodontic research.C57 mice have increasingly become a common choice in inflammation research on dental pulp due to their cost effectiveness, high fertility and vitality. However, the small size of mice and lack of coordination posed significant challenges during experimental procedures. The fixation process is an important factor that higher the technical threshold.We devised a novel mouth gag using readily available materials and simple assemblies to assist researchers in pulp exposure surgery. According to our research, this mouth gag can significantly shorten the time required for beginners to immobilize the mouth and expose the maxillary first molar of mice.

Vorbereitung des Mundknebels für ein Pulpitis-Modell der Maus

Richten Sie zunächst mit der linken Hand einen 8 Mikrometer dicken kieferorthopädischen Bogen aus, während Sie die rechte Hand leicht gegen den Drahtbogen gebeugt halten. Biege mit einer Young-Schlaufenbiegezange die Oberkante des Trapezes etwa acht Millimeter in der Mitte des Bogendrahtes. Stellen Sie sicher, dass sich Punkt A auf der Kante des Zangeschnabels befindet.Halten Sie nun die Zange mit der linken Hand fest. Klemmen Sie das freie Ende des Bügeldrahtes mit dem rechten Daumen und Zeigefinger ein und biegen Sie dann den Bogendraht von Punkt A aus, um einen 120-Grad-Winkel zu bilden. Legen Sie den Bogen auf einen horizontalen Tisch, um zu überprüfen, ob er sich auf einer einzelnen Ebene befindet.Lassen Sie auf jeder Seite des Drahtes etwa neun Millimeter und biegen Sie das freie Ende mit einer Zange in einen 75-Grad-Winkel. Suchen Sie nun Punkt C, der etwa fünf Millimeter von Punkt B entfernt ist.Biegen Sie den Draht an Punkt C, um einen Winkel von 105 Grad zu bilden. Biegen Sie den Draht auf ähnliche Weise an den Punkten D und E. Klemmen Sie die Teile IK und KJ gleichzeitig.Duplizieren Sie die Biegung A, B, C an den Punkten L, J und K, und biegen Sie dann das freie Ende an Punkt J, um es senkrecht zur IKJ-Ebene zu machen. Beugen Sie einen zusätzlichen Zungenspatel für den Unterkieferteil. Klemmen Sie Punkt I fünf Millimeter von Punkt J entfernt und biegen Sie das freie Ende so, dass es sowohl zur IKJ-Ebene als auch zum CD-Teil parallel ist.Lassen Sie als Nächstes einen Abstand von fünf Millimetern zu Punkt I und biegen Sie den Bogen an Punkt H vertikal zu den IH- und JIH-Ebenen. Positionieren Sie Punkt G fünf Millimeter von H entfernt und klemmen Sie die JIHG-Spur fest. Biege dann den Bogen im Punkt G.Biege nun das freie Ende symmetrisch zur KJ-Ebene.Das freie Ende nach Punkt F sollte symmetrisch zum freien Ende an Punkt E sein.Setzen Sie abschließend Gummikappen auf die freien Enden.

preparation-of-the-mouth-gag-for-a-mouse-pulpitis-model

Research

JoVE Journal

Medicine

Preparation of the Mouth Gag for a Mouse Pulpitis Model

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms ...

Forschung

Medizin

To begin, straighten an 8 micron thick orthodontic arch wire with your left hand while keeping the right hand slightly bent against the wire arc. Use a Young loop bending plier to bend the top edge of the trapezoid about eight millimeters at the midpoint of the bow wire. Ensure that point A is on the edge of the plier beak.Now hold the plier with the left hand. Clamp the free end of the bow wire with the right thumb and forefinger, then bend the bow wire from point A to make a 120-degree angle. Place the arch wire on a horizontal table to check if it is on a single plane.Leave about nine millimeters on each side of the wire and use pliers to bend the free end to a 75-degree angle. Now locate point C which is about five millimeters from point B.Bend the wire at point C to make a 105-degree angle. Similarly, bend the wire at points D and E.Clamp the IK and KJ parts simultaneously.Duplicate the A, B, C bending at points L, J, and K, then bend the free end at point J to make it vertical to the IKJ plane. Bend an extra tongue depressor for the mandibular part. Clamp point I five millimeters from point J and bend the free end to make it parallel to both the IKJ plane and the CD part.Next, leave a five millimeter gap from point I and at point H, bend the arch vertically to the IH and JIH planes. Locate point G five millimeters from H and clamp the JIHG lane. Then bend the arch at point G.Now bend the free end symmetrical to the KJ plane.The free end after point F should be symmetrical to the free end at point E.Finally, place rubber caps on the free ends.

Using A Mouth Gag for Pulpitis Assessment in Mice

To begin, fix an anesthetized mouse in a supine position on a fixation plate. Secure the limbs with skin tape. Compress the free ends of a prepared mouth gag with the thumb and the index finger.Fix the mouse's front incisors in the trapezoidal groove between the two arms of the mouth gag. Ensure the arm with the tongue depressor is placed on the mandibula. Check the maxillary first molar to ensure it is free of dental caries, trauma, and odontogenesis.Assess the surrounding gingiva to ensure absence of redness, swelling, or fistula. Then, check the health of the opposite teeth. Next, with a dental burr, drill the occlusal side of the maxillary first molar at 20, 000 RPM.Remove the enamel. With a syringe, drop normal saline every three minutes over the tooth to prevent overheating. Put a number 8 C+file on the lowest position of the drilled pit and pierce the last dentine to expose the pulp chamber.Clean the fragments around the tooth, then remove the mouth gag. The time taken to fix the mouth with the mouth gag and to find the maxillary first molar was significantly shortened relative to the traditional method. The pulp exposure was confirmed through micro CT and reconstruction modeling.At 24 hours post-surgery, most of the pulp tissue retained its morphology.