Name: establishment of a murine pulp exposure model with a novel mouth-gag for pulpitis research
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This study was funded by grants from National Natural Science Foundation of China U21A20368 (L. Y.), 82101000 (H. W.), and 82100982 (F. L.), and by Sichuan Science and Technology Program 2023NSFSC1499 (H. W.). All the original data and images are included in this paper.

The experimental procedures in this study were approved by the ethical committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2021-125). Adult C57BL/6 mice were obtained from Gempharmatech Experimental Animals Company, Chengdu, China. The whole crown of the maxillary first molar erupts 21 days after birth. Mice for surgery should be older than 21 days with normal vitality11. Here, 6 to 8-week-old mice were used for modeling. Figure 1 is a flow diagram showing the protocol used.
1. Preoperative preparation (Figure 2)
<ol>
	<li>Obtain the following instruments: Stereoscopic microscope, fixing plate, medical tape, mouth-gag, minimally invasive dental burr with a diameter of 0.6 mm, dental high-speed dental handpiece, 8# C+ file, heating pad, 1 mL syringe, sterile cotton ball, eye forceps.</li>
	<li>Obtain the following drugs: anesthesia mix, veterinary ointment.</li>
</ol>
2. Preparation of the mouth-gag
<ol>
	<li>Weigh and anesthetize the mouse by intraperitoneal injection of anesthesia mix solution (10% ketamine hydrochloride + 5% xylazine + 85% sterile isotonic saline) at 0.007 mL/g of body weight and confirm proper anesthetization through the toe pinch method. Apply ophthalmic lubricating ointment on the eyes to prevent eyes injury because of desiccation during operation. 
	NOTE: Medical hats, masks, gloves and overalls and other basic protection are necessary. Make sure both surgery environment and mouse chamber are clean and safe. A heat pad for thermal support throughout the procedure is necessary.</li>
	<li>Prepare the mouth-gag as described below (Figure 3).
	<ol>
		<li>Obtain the following materials: Orthodontic arch wire with diameter of 8 &#181;m, young loop bending plier, heavy wire cutter, marker pen, rubber cap with a length of 3 mm and a cross section diameter of 1 mm.</li>
		<li>First, straighten the arch wire with left hand for fixation and thumb, index finger of the right-hand bend slightly against the arc of the wire. Repeat this action several times, it will facilitate the bending to the correct three-dimensional Angle.</li>
		<li>Use the Yong loop bending plier to bend the top edge (Figure 3G, a-i) of the trapezoid (Figure 3C, a-l-k-b) about 8 mm long at the midpoint of the bow wire. Make sure that point a (Figure 3) is on the edge of the plier beak.</li>
		<li>Hold the pliers with left hand, clamp the free end of the bow wire with right thumb and forefinger, and bend the bow wire from point a to make an angle of about 120&#176;. Duplicate the previous action at point i (Figure 3G). Check whether the arch wire is on one plane by putting it on a horizontal table without prying.</li>
		<li>Leave about 9 mm of length on each side (Figure 3D, a-b, l-k) and bend the free-end to a 75&#176; angle by using the same skill as step 2.2.4, while ensuring that each edge is on one plane. Bend this acute angle with the tip of the plier beak.</li>
		<li>Find point c about 5 mm from point b. Follow the same skill to bend a 105&#176; angle on point c. Bend another 105&#176; angle at point d 5mm from point c. Leave about 4.5 mm from point d and find point e. Bend the free end at point e to form an angle of about 100 -105&#176; (Figure 3E). 
		NOTE: The 6-8-week-old C57 mice we used were about 20 g. The spacing of 5 mm could not only jam the upper and lower jaws of the mice without moving, but also would not press the skin of the mice and cause discomfort. If other species or ages of mice are used, please adjust the length of c-d and i-h parts according to the actual situation (Figure 3E,G).</li>
		<li>Bend an extra tongue depressor for mandibular part (Figure 3G, j-i-h-g).</li>
		<li>Duplicate bending steps of a-b-c part on l-k-j part. Clamp i-k and k-j parts at the same time and bend the free end at point j to make it vertical to i-k-j plane. Clamp point i which is 5 mm from point j, bend the free end to make it parallel to both i-k-j plane and c-d part (Figure 3H).</li>
		<li>Leave 5 mm length from point i, at point h, bend the arch vertical to i-h part and parallel to j-i-h plane. Find point g at 5mm from point h. Clamp j-i-h-g plane and bend the free end symmetrical to k-j part. Then free end after point f should be symmetrical to point e-free end (Figure 3H).</li>
		<li>Put rubber caps on the free end (Figure 3F).</li>
	</ol>
	</li>
</ol>
3. Immobilization
<ol>
	<li>Fix the mouse supine on the fixation plate with limbs secured by skin tape. Compress the free ends of mouth-gag with thumb and index finger.</li>
	<li>Fix the mouse front incisors in the trapezoidal groove of two arms. Ensure that the arm with tongue depressor is for mandibula. Adjust the mouth-gag to make sure mouse&#39;s tongue is immobilized but not ischemic.</li>
</ol>
4. Tooth assessment
<ol>
	<li>Ensure the maxillary first molar for surgery should be free of dental caries, trauma and odontogenesis. Ensure there is no redness, swelling, or fistula on the surrounding gingiva. Ensure the opposite teeth are healthy and available to act as the healthy control group.</li>
</ol>
5. Pulp exposure
<ol>
	<li>Use dental burr to drill at the occlusal side of maxillary first molar at a speed of 20,000 rpm. Ensure that the enamel is removed. Keep the operation with dental handpiece only in shallow layer of dentine to prevent excessive thermal stimulation on dental pulp tissue12.</li>
	<li>At the same time, prevent overheating by using a syringe to drop normal saline on the tooth every 3 min during the operation.</li>
	<li>Put an 8# or 10# C+ file on the lowest position of the drilled pit and pierce through the last dentine to expose the pulp chamber. It will be an obvious feeling of falling when local dentine is thoroughly removed. Do not prob too deep, or dental pulp tissue might be brought out of pulp chamber.</li>
	<li>Clean the fragments around the tooth. Take off the mouth-gag; the surgery is finished. Use the opposite maxillary first molar as the control without operation.</li>
</ol>
6. Post-operative care
<ol>
	<li>After surgery, administer carprofen (5 mg/kg) subcutaneously and place the mouse on the chemothermal heating pad in a prone position until recovery from anesthesia. Feed the mice and provide drinking water. The recovery process should be supervised. No other animals should be in the same chamber until the mouse is fully recovered.</li>
</ol>
7. Sample collection and storage
<ol>
	<li>Euthanize the mouse with cervical dislocation under deep anesthetic condition 24 h after operation or any other time point as per experiment9. Cut the skeleton muscles attached to the maxilla and zygoma with ophthalmic scissors. Remove skeleton, frontal bone and soft tissue and take out the lamina gnathostegite with maxillary molars. 
	NOTE: According to He et al., it&#39;s commended that pulpitis sample should be collected less than 72 h after surgery to avoid extensive necrosis in dental pulp tissue13.</li>
	<li>Sagittally divide the gnathostegite in half and store the tissue in 4% paraformaldehyde in PBS, pH 7.4, at 4 &#176;C for 24h-fixation.</li>
</ol>
8. Histological analysis
<ol>
	<li>Wash the tissue with phosphate buffered saline (PBS) and decalcify them in daily-changed decalcification solution of 5% EDTA and 4% sucrose in PBS, pH 7.4, at 4 &#176;C for 2-4 weeks10.</li>
	<li>Embed the 1/2 gnathostegite in paraffin and make sure the sagittal face without teeth is at the bottom of tissue cassettes.</li>
	<li>Cut the paraffin block to 5 &#181;m thick slices with a paraffin microtome. Adjust the angle of the paraffin block according to the proximal, distal, upper as well as lower relationship observed under the microscope to ensure the complete crown pulp and perforation of the first molar can be cut.</li>
</ol>

Creating a Pulp Inflammation Mouse Model Using a Novel Mouth-Gag 

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Authors declare no conflict of interest.

As the solitary soft tissue within teeth, dental pulp fulfills a crucial role in maintaining bioactivity of tooth but remains highly sensitive. The preservation of this vital pulp has become the preferred initial approach in recent endodontic treatments, necessitating a comprehensive understanding of the inflammatory mechanisms of dental pulp16. The spatiotemporal fluctuation of the inflammatory microenvironment and interactions between resident cell types in pulpitis complicates its investigation through in vitro studies11. Instead, in vivo studies offer benefits by replicating the physiological environment found in humans. Utilizing experimental mice, specifically those with overexpressed or knocked down genes, provides an effective instrument for hypothesis validation. The frequently used C57 mice in laboratories, however, pose challenges due to their small size and lack of coordination, rendering the application of stimuli to their teeth problematic17. To address this issue, a comprehensive explanation of novel mouth-gag is needed to assist researchers in performing procedures within the oral cavities of mice. Moreover, this article outlines the protocol for establishing a pulpitis model through pulp exposure on the mice's first molar using the mouth-gag, thereby offering a valuable guide for subsequent research.
After numerous iterations, a scalable mouth-gag that is straightforward to construct was successfully designed. The dimensions and a three-view schematic of the mouth-gag are provided in Figure 3. The protocol significantly simplified the wire bending process by adopting a trapezoidal design in lieu of an arc. The mouth-gag uses a 0.8 mm diameter orthodontic arch wire, which balances the need to prevent slippage from the mouse's mouth and provide sufficient opening force. Moreover, the orthodontic arch wire's elasticity safeguards the temporomandibular joint of the mice. The mouth-gag is compact, resistant to corrosion, and can be stored in a 50 mL centrifuge tube with alcohol for repeated use. Despite a mouse's biting pressure, the mouth-gag remains stable in the mouth, allowing researchers to operate unassisted under a microscope. It should be noted that the mouth-gag's size can be adjusted to fit different mouse body sizes. If the mouth is stretched beyond the mouse's limit, the mouth-gag should be immediately removed to avoid injury to the temporomandibular joint (TMD) or the maxillofacial muscles.
He et al.'s report indicates that necrosis can be detected 24 h post pulp exposure, with the majority of pulp tissue becoming necrotic after 72 h18. Hence, it is essential to collect the pulp tissue within this 72 h window to avoid invalid experimental conclusions due to excessive dead cells. When inserting the C+ file into the pulp chamber, repeated rotation and deep pushing must be avoided to prevent excessive damage to the pulp tissue. If heavy bleeding occurs during the procedure, it is recommended to gently remove the blood using a small cotton ball to prevent coughing. The operation should only be conducted on one side of the mouse maxilla due to the potential negative impact on model accuracy from simultaneous modeling on both sides, which may cause dietary intake complications. Post-surgery, it is advised not to administer anti-inflammatory drugs or antibiotics to the mice.
The mouth-gag, while effective in maintaining the mouse's mouth open, has limitations concerning the surgical site. The mandibular posterior teeth, protected by the tongue pressed with a tongue depressor, are often not clearly visible. Therefore, the procedure is only suitable for operations on the maxilla teeth or mandibular anterior teeth. If the surgery exceeds 20 min, it is recommended to give the mouse a break every 10 min since the mouth-gag's stability depends on antagonizing the mice's occlusal force. C57 mice, chosen for their rapid reproduction and availability, are sensitive to various stimuli; hence, a slight overdose of drugs or stimuli can be lethal. Furthermore, their teeth's small size requires a higher skill level for tissue slicing.
In conclusion, pulp inflammation and necrosis represent pressing challenges in pulp regeneration. This study offers a comprehensive demonstration of creating a pulpitis model in mice, with immunofluorescence results verifying the inflammatory factors. This article proposes a novel, convenient mouth-gag design, which provides the operator with unobstructed sight by keeping the mouse's mouth open without tongue interference. However, operations on the mandibular posterior teeth remain a challenge. Considering the advantages of using experimental mice, endodontic modeling in mice holds significant promise, and it is planned to anticipate further reductions in the technical threshold.

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.

<table><tbody><tr><td>&lt;strong&gt;Animal&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C57/B6J mice</td><td>Gempharmatech Experimental Animals Company</td><td>C57/B6J</td><td>For the establishment of pulp exposure</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 mL syringe</td><td>Chengdu Xinjin Shifeng Medical Apparatus &amp;amp; Instruments Co. LTD.</td><td>SB1-074(IV)</td><td>Apply in drug injection.</td></tr><tr><td>8# C+ file</td><td>Readysteel</td><td>0010047</td><td>Apply in exposing the roof of pulp chamber.</td></tr><tr><td>Anesthesia Mix solution</td><td></td><td></td><td>10% ketamine hydrochloride+ 5% xylazine + 85% sterile isotonic saline.&amp;nbsp;</td></tr><tr><td>DAPI Staining Solution</td><td>Beyotime</td><td>C1005</td><td>Apply in immunofluorescence staining for counter-staining of nucleus.</td></tr><tr><td>Dental high-speed dental handpiece</td><td>Jing yuan electronic commerce technology</td><td>WJ-422</td><td>Apply in pulp exposure.</td></tr><tr><td>Heavy wire cutter</td><td>Jirui Medical Instrument Co., Ltd.</td><td>none</td><td>Apply inarc cutting.</td></tr><tr><td>Hematoxylin and Eosin Stain kit</td><td>Biosharp</td><td>BL700B</td><td>For the histological analysis of the slides.</td></tr><tr><td>IL-6 antibody</td><td>Novus</td><td>NBP2-89149</td><td>Apply in immunofluorescence staining to detect the inflammation of the dental pulp.</td></tr><tr><td>Ketamine(Ketamine hydrochloride)</td><td>Vet One, Boise, Idaho, USA</td><td>C3N VT1</td><td>100mg/kg, IP. Apply in nesthetization.</td></tr><tr><td>Medical tap</td><td>3M</td><td>1530</td><td>Apply in mice immobilization.</td></tr><tr><td>Orthodontic arch wire&amp;nbsp;</td><td>Shanghai Wei Rong Medical Apparatus Co. LTD.</td><td>K417</td><td>Diameter of 8&amp;micro;m</td></tr><tr><td>Round dental burr (0.6 mm)</td><td>Shofu global</td><td>072208</td><td>Apply in removing enamel and shallow layer of dentin.</td></tr><tr><td>Young loop bending plier</td><td>Jirui Medical Instrument Co., Ltd.</td><td>none</td><td>Apply in arc bending.</td></tr></tbody></table>

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.

The procedure described above was performed on the right maxillary first molar of 3, 6-8 weeks old C57BL/6 mice, while the left maxillary first molars were preserved as control. Histology and immunofluorescence results from blank control, 12 h pulpitis and 24 h pulpitis samples were utilized for demonstration.
Following protocol of CT analysis from Goldman et al.15, pulp exposure was confirmed through micro-CT and reconstruction modeling in Figure 4 A-C. Sagittal slices of the maxillary first molars, both from the control and surgery sides, underwent HE staining (Figure 5). Pulp tissue necrosis and cell morphology disintegration at the wound margin were shown. The necrosis was mainly concentrated in the pulp tissue near the perforation, and the shape of the pulp tissue on the unopened side was normal. At 24 h, most of the pulp tissues, including the root pulp, were morphologically intact. (Figure 5).
The expression of IL-614 was low in control, and a small amount of IL-6 could be observed around the wound at 12 h, while the expression of IL-6 was significantly increased at 24 h. Moreover, the expression of IL-6 was mainly concentrated in the wound margin and the middle pulp horn (Figure 6). In Figure 6 D,E, number of IL-6+ dental pulp cells and ratio of IL-6+ cells to total dental pulp cells increases over time in three time points. It can be considered that the area gradually develops inflammation and aggravates after pulp exposure.
We invited 5 colleagues who have never performed surgery on maxillary teeth of C57 mice to calculate their time needed to immobilize the upper and lower jaw of mouse (Stage 1) and expose mouse maxillary first molar (Stage 2) following the traditional procedure with two rubber band referring to the protocol published by Goldman et al. or our protocol with the mouth-gag15. Time for placing the drill correctly on the mouse maxillary first molar under microscope was also calculated for analysis. The results in Figure 3 J,K suggested that the time of mouth fixation and finding mice maxillary first molar with the mouth-gag were significantly shortened compared with the traditional way(P&#60;0.05). The use of mouth-gag can improve operation efficiency and reduce operation difficulty.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66016/66016fig01.jpg" /> 
Figure 1: Flow diagram of pulp exposure procedure. (A) After fixation with mouth-gag, the maxillary first molar of the mouse should be fully exposed under the microscope. (B) Use a high-speed dental handpiece with minimally invasive dental burr to remove occlusal enamel and superficial dentin of the first molar but be careful not to penetrate the dentin directly to avoid excessive influence on the pulp caused by overheating. (C) Use 8# C+ file to penetrate the remaining dentin and expose the pulp. (D) Samples were collected 24 h after surgery. HE and immunofluorescence staining proved that a pulpitis model could be established at this time point. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66016/66016fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66016/66016fig02.jpg" /> 
Figure 2: Equipment for pulp exposure procedure. (A) Stereoscopic microscope and motor of dental high-speed dental handpiece. (B) The 8# C+ file, minimally invasive dental burr, mouth-gag, tweezers, and dental high-speed dental handpiece. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66016/66016fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66016/66016fig03v2.jpg" /> 
Figure 3: Making the mouth-gag. (A) Bend the wire to make two working arms of the mouth-gag. (B-C) Steps of bending tongue spatula for mandible. (D-E) No tongue spatula for maxilla. (F, H) Rubber caps or bent ends are needed to protect injury from being pricked. (G) Three views of the mouth-gag for reference. (I) Clinical use of mouth-gag. (J) The average time for beginners to fix the upper and lower jaws (Stage 1) using traditional fixation method and mouth-gag, respectively. (K) The average time for beginners to clearly find the maxillary first molar (Stage 2) using traditional fixation method and mouth-gag, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66016/66016fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66016/66016fig04.jpg" /> 
Figure 4: Micro-CT analysis of operated fist molar with perforation marked by red dotted line. (A) Sagittal plane of tooth, ensure that no perforation on pulp-chamber floor exists. (B) Coronal plane. Corresponding to the perforation in (A), complete penetration of the enamel dentin circled with red dotted line can be observed. (C) Occlusal plane of CT reconstruction model. The location of perforation circled with red dotted line is confirmed in a more intuitive way and the floor of the pulp chamber can be observed through perforation. (D-G) Intraoperative photos of the treatment. (D) Check the maxillary first molar of the mouse to ensure there are no cavities or malformation. (E) Using a minimally invasive burr to remove enamel and shallow dentin. As can be seen in the image, a pit (circled with dashed white line) can be observed on the occlusal face of the tooth without perforation. (F) Using 8# C+ file to penetrate the remaining dentin, the file can get stuck in the tooth without hand support. (G) When the file is removed, the tooth has a pink perforation, indicating successful pulp exposure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66016/66016fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66016/66016fig05.jpg" /> 
Figure 5: Hematoxylin-eosin staining. (A) Holistic view of untreated first molar for control. (A-1,2,3) High-magnification figures corresponding to 1, 2, 3 in panel (A). The shape of dental pulp tissue at 3 positions were intact, and odontoblasts were in an orderly arrangement. (B) Holistic view of teeth 12 h after surgery. (B-1,2,3) High-magnification figures corresponding to 1, 2, 3 in panel (B). The shape of dental pulp tissue at position 1 and 2 were generally intact. Necrosis could be observed near the perforation. (C) Holistic view of teeth 24 h after surgery. (C-1,2,3) High-magnification figures corresponding to 1, 2, 3 in panel (C). Necrosis extends from a single pulp horn to nearby pulp tissue. But most of the pulp tissues, including the root pulp are morphologically intact. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66016/66016fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66016/66016fig06v2.jpg" /> 
Figure 6: Immunofluorescence staining. (A) Holistic view of untreated first molar for control. (A-1,2,3) High-magnification figures corresponding to 1, 2, 3 in panel (A). Nearly no expression of IL-6 could be observed. (B) Holistic view of teeth 12 h after surgery. (B-1, 2, 3) High-magnification figures corresponding to 1, 2, 3 in panel (B). Increasing IL-6 concentrated in tissue near the perforation shown in B-3. No obvious changes were observed in B-1,2. (C) Holistic view of teeth 24 h after surgery. (C-1, 2, 3) High-magnification figures corresponding to 1, 2, 3 in panel (C). Expression of IL-6 was significantly increased in C-2,3. (D) Total amount of IL-6+ dental pulp cells in control, 12 h pulp exposure, 24 h pulp exposure immunofluorescence staining results. (E) Ratio of IL-6+ cells to total amount of dental pulp cells in control, 12 h pulp exposure, 24 h pulp exposure immunofluorescence staining results. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66016/66016fig06largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research at JoVE.com

Author Spotlight: Enhancing Dental Pulp Research with Improved Mouse Models

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

921 Views.  Sichuan University. Individuele experimenten zoals 2D- of 3D-celculturen zijn op grote schaal gebruikt om de reacties van tandpulpacellen te testen. Deze experimenten kunnen de interactie tussen pulpaweefsel en het systemische immuunsysteem niet weerspiegelen. Daarom worden in vivo experimenten steeds populairder in endodontisch onderzoek.C57-muizen zijn steeds meer een veel voorkomende keuze geworden in onderzoek naar ontstekingen op tandpulp vanwege hun kosteneffectiviteit, hoge vruchtbaarheid en vitali...

Video: Auteur in de schijnwerpers: onderzoek naar tandpulp verbeteren met verbeterde muismodellen

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Immunology and Infection

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1&#946;, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1&#946; levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation.

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, ...

Experimental Model of Ligature-Induced Peri-Implantitis in Mice

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is characterized by inflammation in the tissues around dental implants with progressive loss of supporting bone. To optimize dental implants' longevity in terms of health and functionality, it is crucial to understand the peri-implantitis pathophysiology. In this regard, using mouse models in research has proven clear benefits in recreating clinical circumstances. This study aimed to describe an experimental model of ligature-induced peri-implantitis in mice and determine whether there is effectiveness in inducing this disease, given the observed bone and tissue changes. The experimental peri-implantitis induction comprehends the following steps: teeth extraction, implant placement, and ligature-inducted PI. A sample of eighteen 3-week-old C57BL/6J male mice was divided into two groups, ligature (N=9) and control non-ligature (N=9). The evaluation of clinical, radiographical, and histological factors was performed. The ligature group showed significantly higher bone loss, increased soft tissue edema, and apical epithelial migration than the non-ligature group. It was concluded that this pre-clinical model can successfully induce peri-implantitis in mice.

This is a report on an experimental model of ligature-induced peri-implantitis in mice. We describe all surgical steps, from pre- and post-operative management of the animals, extractions, implant placement, and ligature-induced peri-implantitis.

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Biology

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone regeneration and intramembranous ossification. C57/J6 mice were used to extract the mandibular first molar to establish this model. They were euthanized, and the bilateral mandibles harvested, at 1 week and 4 weeks post-surgery, respectively. Subsequent serial stereoscopic harvest, histological assessment, and immunofluorescence staining were performed to demonstrate successful surgery. Immediately after surgery, the stereoscopic images displayed an empty extraction socket. The hematoxylin and eosin (H&#38;E) at 1 week and Masson staining at 4 weeks post-surgery showed that the area of the original root was partially and fully filled with bone trabeculae, respectively. The immunofluorescence staining showed that, compared with the homeostasis side, the Sp7 expression increased at 1 week post-surgery, suggesting vigorous osteogenesis in the alveolar fossa. All these results demonstrated a practicable murine tooth extraction socket healing model. Upcoming studies revealing the mechanisms of jawbone defect healing or socket healing could adopt this method.

This protocol demonstrates step-by-step details of how to extract the mandibular first molar in the mouse. It provides an alternative method for researchers focusing on jawbone healing and regeneration.

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone ...

Citric Acid-Induced Mouse Model for Cough Hypersensitivity

Establishment of a Mouse Model with Cough Hypersensitivity via Inhalation of Citric Acid

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable economic burden. Increased cough sensitivity is a pathophysiological hallmark of chronic cough. It has been observed that cough hypersensitivity is related to airway inflammation, remodeling of airway sensory nerves, and alterations in the central nervous system. However, the precise molecular mechanisms remain unclear and require further elucidation using suitable animal models. Previous studies have utilized guinea pigs as models for studying cough, but these models present several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. In addition, guinea pigs typically exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. In this study, we established a mouse model with cough hypersensitivity&#160;via&#160;continuous inhalation of citric acid (CA). This model is straightforward to operate and yields reproducible results, making it a valuable tool for further studies on the mechanisms and potential novel treatments for chronic cough.

This protocol describes the development of a mouse model with cough hypersensitivity, which can serve as an ideal model for studying the mechanisms of chronic cough.

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable ...

Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

Source: Barkley, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60809">A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.</a>&#160;J. Vis. Exp. (2020).
This video showcases a technique for establishing a co-culture of trigeminal neurons and primary dental pulp cells using a transwell filter. In this physically separated condition, mesenchymal cells obtained from dental pulp tissue secrete paracrine signaling molecules that traverse through the pores, promoting the differentiation and migration of neurons towards the mesenchymal cells establishing a co-culture system.

1. Plate Preparation
NOTE: Be sure the plate lid is on during all incubation and rinsing steps outside of the sterile tissue culture hood to prevent ...

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

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