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>
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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 ...

Our protocol enables the study of periapical periodontitis in an accessible and controlled mouse model. Therefore, this protocol has advantages over patient samples or in-vitro models. The main advantages of this technique are that the mouse has been well studied and that the method is technically simple in terms of facility requirements and is cost effective.As this procedure is challenging due to the small dimensions of the teeth, it is beneficial to visualize the procedure to learn about the positioning and performance of the technique. After confirming a lack of response to toe pinch, place the mouse on the dominant hand side on a closed-cell extruded polystyrene foam surface and secure the paws with tape. Using a single rubber band per pair of incisors, and long needles pined to the closed-cell extruded polystyrene foam surface, prop the mouth open.Use closed forceps taped to the foam to retract the right cheek. And place the foam and mouse under a microscope and a light source. Then, retract the tongue using a dental spatula and use a 27 gauge needle to inject 25 to 50 microliters of local anesthetic into the mucobuccal fold adjacent to the first mandibular molar.Tissue swelling around the injection site should be observed. To expose the pulp, use any suitable dental motor equipped with a round, high speed diamond 0.8 millimeter dental bur at a speed of 800 rounds per minute, to drill the occlusal part of the first right mandibular molar until the pulp horns are visible through the dentin. Use a K or H file, number eight or number 10 to pierce the pulp and break the dentin covering the mesial and distal pulp horns to allow insertion of the file into the horns.Continue working with the files inside the pulp as deeply as possible while widening the openings with the files, bleeding from the pulp is typically observed round the sharp edges of the tooth with the bur and remove the tooth from the aclusion to reduce pain during the experiment. Use a micro brush to clean any debris. And leave the contralateral left first mandibular molar as a control.For the first three days after the procedure, weight the animals and inject 0.1 milligrams per kilogram of buprenorphine intraperitoneally once a day. Continue to monitor the animals over the next 42 days by weighing and general behavioral assessment two to three times a week. And deliver food pellets softened with water in a Petri dish on the floor of the cage throughout the course of the follow-up period.At the end of the experiment, use surgical scissors and forceps to collect the part of the jaw that includes the three molars on both the treated and non-treated sides. And use forceps to peel off as much of the soft tissue as possible, leaving mostly bone and teeth on the samples. Rinse the tissue briefly in PBS.Before fixation in four per cent paraformaldehyde for 24 to 48 hours. Then rinse the samples three times in fresh PBS. For Micro-Computed Tomography, or Micro-CT analysis place the harvested tissues in a 12 millimeter diameter tube containing 1.5 milliliters of PBS on the scanner stage.With the samples oriented so that the buccal or lingual surfaces of the tooth and root lay parallel to the bottom of the tube. Use a sponge to separate the samples. And cover the top of the tube with flexible film.Select the control file, and set the energy to 70 kilovolts, the intensity to 114 micro amps, and the resolution to a six micrometer cubed Voxel size. Click Scout-View. When the image appears, click Reference-Line and mark the area of the samples for scanning, clicking Add Scan after each sample.When all of the samples have been marked, go to the task list, and select Start Interact Tasks. For contouring of the Micro-CT slices, select the slice that represents the approximate middle of the tooth, in which the coronal pulp, radicular pulp of both canals, and both apical foramens are present. Click the contour panel, and starting from the distal point of the distal root apex, mark the apical border coronally to the radial paque apical area, through the mesial border of the mesial root apex, following the distal border of the mesial root, and the mesial border of the distal root, through the furcation region.Copy and paste the resulting contour, and adjust the contour accordingly to five slices positioned on either side of the middle slice. To calculate the tissue volume of the contour marked for each sample, under Micro-CT Evaluation, select Task and click 3D-Evaluation. Then, in the window of 3D-Evaluation, click Select and select a filter to calculate the tissue volume.At the end of the Micro-CT analysis, transfer the tissues from the scanner to a micro centrifuge tube containing one milliliter of EDTA for 10 days. At the end of the decalcification, place the samples into histological cassettes containing increasing ethanol concentrations, for one hour per concentration. After the second ethanol immersion, transfer the samples into xylene for two one-hour immersion, Before transferring the cassettes into 60 degree Celsius liquid paraffin in a chemical hood overnight to allow the xylene to evaporate.The next morning, place the dehydrated samples with the crown and roots oriented parallel to the bottom of the mold, in a histological blocking machine to embed the samples in paraffin. To obtain sections, secure the paraffin block sample onto the microtone cutting block, and trim the sample until the tissue of interest is reached. When any part of the tooth is visible, begin obtaining six micrometer thick sections, adjusting the cutting angle until sagittal sections that include the coronal and radicular pulp and the apical foramen are obtained.Here, a whole treated mandible is shown. Magnification of the treated first right molar, allows observation of the exposure of both the mesial and distal pulp horns and the entrance to the canals. Micro-CT imaging allows visualization of the mesial pulp exposure, and the mesial and distal periapical radiolucent areas of bone resorbtion.Indeed, a significant periapical bone resorbtion of the tissue volume is measured in the treated teeth, compared to the controls. Histological analysis indicates that in the treated tooth, the dental pulp itself presents necrosis, which can be observed clearly after H and E staining compared to the control organized pulp tissue. Additionally, and importantly, in the periapical region of the treated tooth, a periapical lesion composed of immune cells is observed, that is absent in the control teeth.It is vital to position the mouse correctly during the pulp exposure as a correct positioning enables a good access to the mouth of the animal and optimal results. Additional methods of periapical inflammation analysis such as immunohistochemical tissue staining and fax and detailed molecular analysis of the cells can be performed following this protocol. This protocol is significant, not only for dental research, but also for immunological research, as it provides a model for local induced inflammation with a built-in internal control.

Research

JoVE Journal

Biology

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Terminal H-reflex Measurements in Mice

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS).
In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits ...

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...