Name: inducing apical periodontitis in mice
Uploaded: 2019-08-06T13:30:00
Description: Watch this Scientific Journal Video about Inducing Apical Periodontitis in Mice at JoVE.com

We would like to acknowledge Dr. Oded Heyman for his help with animal positioning, Raphael Lieber for help with micro-CT analysis, and Prof. Andiara De Rossi Daldegan for advice on preforming the experiment. We would also like to acknowledge Dr. Sidney Cohen for critical reading and editing.
This work was supported by a grant from the Dr. Izador I. Cabakoff Research Endowment Fund to MK and IA, and a Yitzhak Navon fellowship from the Israel Ministry of Science and Technology to EG.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of The Hebrew University (Ethics no. MD-17-15093-5).
1. Animal anesthesia and positioning
<ol>
	<li>Prepare sterile solutions as described below.
	<ol>
		<li>Prepare 5 mL of 7 mg/mL of Ketamine and 0.09 mg/mL Medetomidine diluted in Phosphate Buffer Solution (PBS)/Saline.&#160;</li>
		<li>Prepare a sterile solution of Atipamezole (0.4 mg/mL) diluted in PBS/Saline (recommended to prepare a...

<ol>
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J., Kiss, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+protective+and+destructive+regulatory+pathways+in+apical+periodontitis.">Overlapping protective and destructive regulatory pathways in apical periodontitis.</a> Journal of Endodontics. 40 (2), 155-163 (2014).</li><li>Graunaite, I., Lodiene, G., Maciulskiene, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+apical+periodontitis:+a+literature+review.">Pathogenesis of apical periodontitis: a literature review.</a> Journal of Oral and Maxillofacial Research. 2 (4), e1 (2012).</li><li>Hussein, F. E., Liew, A. K., Ramlee, R. A., Abdullah, D., Chong, B. 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M., Sasaki, H., Hirai, K., Andrada, A. C., Gomes-Filho, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+hypertension+and+periapical+lesion:+an+in+vitro+and+in+vivo+study.">Relationship between hypertension and periapical lesion: an in vitro and in vivo study.</a> Brazillian Oral Research. 30 (1), e78 (2016).</li><li>Rao, N. J., Wang, J. Y., Yu, R. Q., Leung, Y. Y., Zheng, L. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+animal+models+for+rheumatoid+arthritis.">Experimental animal models for rheumatoid arthritis.</a> Immunopharmacology and Immunotoxicology. 40 (3), 193-200 (2018).</li><li>Shah, 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=Clastic+cells+are+absent+around+the+root+surface+in+pulp-exposed+periapical+periodontitis+lesions+in+mice.">Clastic cells are absent around the root surface in pulp-exposed periapical periodontitis lesions in mice.</a> Oral Disease. 24 (1-2), 57-62 (2018).</li><li>Wan, C., 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=MMP9+deficiency+increased+the+size+of+experimentally+induced+apical+periodontitis.">MMP9 deficiency increased the size of experimentally induced apical periodontitis.</a> Journal of Endodontics. 40 (5), 658-664 (2014).</li><li>Bezerra da Silva, R. 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A method is introduced here for the induction of apical periodontitis in mice. The goal of the method is to exploit the apical periodontitis condition for studying mechanisms and consequences of this inflammatory process. Apical periodontitis was induced in 6-8 week old mice, an age in which the roots are fully developed24. In order to cause apical periodontitis in this model, the tooth pulp of mouse mandibular molars is exposed using a dental burr. Bacteria from the oral flora of the...

The goal of this method is to induce apical periodontitis in a mouse by contaminating the apex with the natural microflora, and to then study various characteristics of this pathological process.
Apical periodontitis (AP) is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. This dental disease can cause severe pain and must be treated by a dentist. The treatment options include root canal treatment (primary or secondary), endodontic surgery, tooth extraction, or follow-up depending on the clinical and radiographic findings, and the opinion of the clinician. The mechanism of this inflammatory process, although studied for several decades1,2,3, is still not comprehensively understood. Considering the severity of this pathology, there is thus a clear need for research addressing its fundamental nature. Thus, systems where the study of AP is possible are of great scientific interest.
Since AP is a complex pathological process involving the local tissues and the immune system, in vitro studies are insufficient for a complete understanding of the processes. Study of clinical samples of this disease are also problematic due to ethical limitations and significant variability between different people and different clinical stages4,5, and hence the necessity of in vivo models. These models are based on the concept of exposing the dental pulp to contamination and observing the inflammatory reaction of the body to this stimulus in the periapical tissues6,7. Common in vivo models include rodents or larger animals such as dogs. Despite the clinical challenge in treating mice, which are very small animals with miniature teeth, the advantages of the mouse model are significant: practically, working with mice is technically simple in terms of facilities and is most cost effective, and scientifically, the mouse is a well-studied animal model with readily available genetic and molecular tools and a well-studied genome. Indeed, previous studies used a mouse model for studying inflammatory and bone resorption signals and cells involved in apical periodontitis8,9,10,11. Therefore, a clear protocol on how to use a mouse model for the study of AP is needed. Here, we describe such a protocol.
The protocol described here is has the big advantage of being appropriate to study knock-out (KO) mice and learn how the lack of a specific gene affects dental inflammation7,12. Other useful applications of this protocol include the study of effects of medications and systemic conditions on the development of apical periodontitis13, the effect of apical periodontitis on the development of osteonecrosis of the jaws14,15 and stem cell therapy for bone regeneration16.
This protocol can also be generalized as a model to study local inflammation. To study the inflammatory process, several mouse models have been developed, which include for example induced colitis or arthritis17,18. These models have systemic effects and have no built-in control in the same animal. Models for induced apical periodontitis, which include a contralateral control without inflammation, have the advantage of overcoming these limitations14,19.
The protocol described below is therefore useful for researchers who are interested in local inflammatory processes. The controlled nature of this inflammation, its confinement to a specific location, and the contralateral control tooth, all make this protocol valuable for studying the mechanisms involved in this process. Moreover, the protocol is useful for researchers interested in the clinical aspects of periapical inflammation. The mouse model is ideal to study different variables of the disease, in addition to the advantage of being able to easily perform genetic manipulations in the mouse model, to investigate the activity of specific genes in periapical inflammation.
Technically, the clinical procedure is challenging to carry out due to the small dimensions of the mice teeth. It will be beneficial to visualize this procedure in order to learn about positioning, equipment needed, and performance.

<table>
	<tbody>
		<tr>
			<td>Atipamezole hydrochloride</td>
			<td>Eurovet Animal Health</td>
			<td>CAS 104075-48-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ATR</td>
			<td>dentsply</td>
			<td>tecnika</td>
			<td></td>
		</tr>
		<tr>
			<td>blocking machine</td>
			<td>Leica</td>
			<td>EG1150H</td>
			<td></td>
		</tr>
		<tr>
			<td>buprenorphine</td>
			<td>vetmarket</td>
			<td>163451</td>
			<td></td>
		</tr>
		<tr>
			<td>clinical microscope/binocular</td>
			<td>Olympus</td>
			<td>Sz61</td>
			<td></td>
		</tr>
		<tr>
			<td>dental bur</td>
			<td>Komet dental</td>
			<td>ZR8801L 315 008</td>
			<td></td>
		</tr>
		<tr>
			<td>dental spatula</td>
			<td>Premier</td>
			<td>1003737</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>J.T Baker</td>
			<td>8993</td>
			<td></td>
		</tr>
		<tr>
			<td>entelan</td>
			<td>mercury</td>
			<td>1.07961</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y solution, alcoholic</td>
			<td>SIGMA</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>hematoxylin solution, Mayer&#39;s</td>
			<td>SIGMA</td>
			<td>MHS 16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Vetoquinol</td>
			<td>CAS 1867-669</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine hydrochloride (cepetor)</td>
			<td>CP-pharma GmbH</td>
			<td>CAS 86347-15-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mepivacaine HCl 3%</td>
			<td>Teva</td>
			<td>CAS 96-88-8</td>
			<td></td>
		</tr>
		<tr>
			<td>microbrushes- adjustable precision applicators</td>
			<td>PARKELL</td>
			<td>S379</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-ct scanner</td>
			<td>scanco</td>
			<td>uCT 40</td>
			<td></td>
		</tr>
		<tr>
			<td>parafin</td>
			<td>Leica</td>
			<td>39602004</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>SIGMA</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>EMS</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol eye ointment (5%)</td>
			<td>Rekah pharmaceutical</td>
			<td>CAS 56-75-7</td>
			<td></td>
		</tr>
		<tr>
			<td>tweezers</td>
			<td>WAM</td>
			<td>Ref-CT</td>
			<td></td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>Eurovet Animal Health</td>
			<td>CAS 7361-61-7</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Gadot</td>
			<td>CAS 1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A flow chart of the experimental steps is presented in Figure 1. As mentioned in the protocol, the mice are anesthetized, and their first mandibular molar on one side is drilled until pulp exposure, while the contralateral tooth is left as a control. Next, the teeth are left to be contaminated by the oral flora for 42 days, during which they are monitored and receive pain medication. After 42 days, mice are euthanized, and the teeth and adjacent jaw are taken...

Watch this Scientific Journal Video about Inducing Apical Periodontitis in Mice at JoVE.com

Inducing Apical Periodontitis in Mice

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 ...

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