Inducing Apical Periodontitis in Mice

Podsumowanie

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse'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.

Streszczenie

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.

Wprowadzenie

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 decades^1,2,3, 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 stages^4,5, 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 tissues^6,7. 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 periodontitis^8,9,10,11. 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 inflammation^7,12. 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 jaws^14,15 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 arthritis^17,18. 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 limitations^14,19.

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.

Protokół

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

1. Prepare sterile solutions as described below.
   1. Prepare 5 mL of 7 mg/mL of Ketamine and 0.09 mg/mL Medetomidine diluted in Phosphate Buffer Solution (PBS)/Saline. 
   2. Prepare a sterile solution of Atipamezole (0.4 mg/mL) diluted in PBS/Saline (recommended to prepare an amount of 5 mL).
   3. Prepare a sterile solution of Mepivacaine (7.5 mg/mL) diluted in PBS/Saline for local anesthesia (one supplied vial of Mepivacaine is sufficient for a stock of 7.2 mL).
2. Use 6-8 weeks old female C57BL/6 mice for the experiment. Keep animals in a specific pathogen free (SPF) animal unit, with standard water and food provided by the facility and the animal feeding ad libitum.
3. Weigh mice and inject intraperitoneally (IP) a volume of 10 µL for each gram of the weight. For example, for a mouse that weighs 20 grams,
   inject 200 µL of Ketamine/Medetomidine solution. This will give a final concentration of (70 mg/kg) Ketamine and (0.9 mg/kg) Medetomidine
   for each animal.
4. In order to prevent eye ulceration due to dryness during the procedure, apply eye ointment containing Chloramphenicol (5%) on the animals' eyes while they are under anesthesia. Keep the animals warm with a lamp while they are anesthetized. Check depth of anesthesia by checking pedal reflex (performing firm toe pinch).
5. After the mouse is anesthetized, position the mouse laying on its right side (for a right handed researcher) on a closed-cell extruded polystyrene foam surface and attach feet to the surface using tape.
6. Open the mouth by using small rubber bands around the incisors (1 for upper incisors, and 1 for lower incisors) held in place by long needles pinned into the closed-cell extruded polystyrene foam surface.
7. Retract the right cheek by using forceps taped to the surface. Use forceps that are closed in steady state.
8. Place the surface with mouse in a comfortable working position enabling access to mandibular molars, using proper magnification (a binocular microscope or clinical microscope) and light.
9. Retract the tongue by using forceps or dental spatula and inject local anesthesia with a 27 Gauge needle into the mucobuccal fold adjacent to the first mandibular molar. 25-50 µL is sufficient. Swelling of the tissue around the injection site should be visualized.

2. Pulp exposure

1. Use a 1/4 round dental burr, or a same size diamond burr (a long shank is recommended) at a speed of 800 rounds per minute (rpm), attached to any suitable dental motor (for example an automatic torque reduction (ATR) motor).
2. Drill on the occlusal part of the first right mandibular molar until the pulp horns are visible through the dentin. use short bursts of the motor to prevent area over-heating.
3. Use a K- file or H-file #8 or #10 to pierce the pulp and insert the file into the pulp horns (mesial and distal) by breaking the dentin covering them. Files are sterilized by autoclave.
4. Continue working with the files inside the pulp as deep as possible, while widening the openings with the files. Usually there will be bleeding visible from the pulp.
5. Make sure to round sharp edges of the tooth with the burr and remove the tooth from occlusion, in order to reduce pain during the experiment. Clean debris during the procedure with a microbrush.
6. Leave the contralateral tooth (left first mandibular molar) as a control.

3. End of clinical procedure

1. Release mouse from the fixation state and inject Atipamezole IP (10 µL for each gram of body weight. This will give a final dose of (4 mg/kg) Atipamezole.
2. Inject buprenorphine diluted in PBS/saline (0.05 mg/kg) IP (recommended to prepare an amount of 3 mL on the day of the procedure). See Discussion for explanation why pain relief is necessary.
3. Make sure the animals recover from anesthesia before leaving them. Give them soft food because they may be incapable of eating hard food due to tooth pain.

4. Post procedure follow-up (42 days)

1. For the first 3 days after the procedure, weigh the animals and inject Buprenorphine (0.1 mg/kg) IP once a day (recommended to prepare an amount of 6mL).
2. During the time of the experiment (42 days when inflammation builds up) monitor the animals by weight and general behavioral assessment 2-3 times a week.
3. Deliver soft food to the animals during the course of the follow-up period. Wet the food with water and put it in a petri-dish on the floor of the cage.

5. Experiment termination and analysis

1. When 42 days¹¹ of follow-up have been completed , anesthetize animals IP with ketamine/xylazine at a concentration of 106.25 mg/kg Ketamine, and 75 mg/kg Xylazine (A stock solution of 42.5 mg/mL Ketamine, and 1.5 mg/mL Xylazine in a total volume of 2 mL is recommended) and preform cervical dislocation.
   NOTE: There are different options for possible analyses in order to evaluate inflammation^20,21. Here the analysis of the tissue by micro-CT and histological staining will be described.
2. After euthanasia, use surgical scissors and forceps to cut out part of the jaw including the 3 molars (separately for each side- treated and non-treated control). By using forceps peel off as much as the soft tissue as possible, leaving mostly bone and teeth on the sample.
3. Rinse the tissue briefly in PBS, and then put it in paraformaldehyde (PFA) 4% (diluted in PBS) for 24-48 hours for fixation.
   CAUTION: Use PFA in a chemical hood, and according to its Material Safety Data Sheet (MSDS) guidelines.
4. After 24-48 hours, rinse with PBS 3x in order to wash out the PFA.
5. Micro-CT
   1. For micro-CT analysis, take the harvested tissues (part of the mandible including 3 molars, in dimensions of approximately 4 mm x 5 mmx 1 mm) to a micro-CT scanner, place them in an 12 mm diameter tube filled with 1.5 mL of PBS oriented so that the buccal or lingual surfaces of the tooth and root are laying parallel to the bottom of the tube. Separate between the samples with sponge, and cover the top of the tube with flexible film.
   2. Open the scanning panel and choose the measurement number. Choose the control file with the following parameters: energy of 70 kV, intensity of 114 µA, and resolution of 6 µm³ voxel size.
   3. Click on scout view, and when the image appears, click reference line and mark the area of the samples for scanning. After each sample click add scan. When done marking the samples, go to the task list and choose start interact tasks.
      NOTE: The same samples can subsequently be used for histology. It is important that they do not dry out during the CT-scan.
6. Use an appropriate computer program for micro-CT alignment and analysis.
   1. Open the sample on the XY plane in the micro-CT evaluation. Choose three points on each sample for alignment:
      The mesial apical constriction - defines the origin of the coordinate system.
      The coronal part of the mesial canal - defines a point on the Z axis.
      ​The distal apical constriction - defines a point on the XZ plane.
   2. Use the measurement tool on the left panel, and draw a line reaching each point. Define each of the three points by an X, Y & Z value, as it appears in the right panel.
   3. Define the area of interest in the sample by choosing task under µCT evaluation, and click on 3D evaluation. A rectangle and a window with the dimensions per axis will appear on the screen. Match the dimensions of the rectangle to fit the area of interest on the XY axis by clicking in the corners with the middle button of the mouse. Choose the dimensions in the Z axis by inserting the first slice number of interest in the Z square under VOI Start, and the number of slices from the first until the last slice number of interest in the Z square under Dim.
   4. Click on applications under session manager, and choose DECterm. Wait a few seconds for a window to open, and then type the five steps of slanted script described below according to the following instructions (between the lines click enter):
      ipl
      isq_to_aim
      aim1
   5. Copy the isq (file of interest) name: choose views under session manager and click microCTdata. Find the file being working on, and middle click on the file. Then middle click back in the window of the DECterm.
   6. Enter the X, Y & Z values (with a space between them) which were determined under VOI when choosing region of interest.
   7. Enter the X, Y & Z values (with a space between them) which were determined under dim when choosing region of interest.
   8. Wait until receiving a message: isq to aim completed.
      (step II- alignment):
      align z
      aim1
      aim2
   9. Enter the X, Y & Z values (with a space between them) which were determined for the origin of the coordinate system.
   10. Enter the X, Y & Z values (with a space between them) which were determined for the point on the z axis.
   11. Enter the X, Y & Z values (with a space between them) which were determined for the point on the XZ plane.
   12. Wait until receiving a message: "align_Z completed"
       (step III- header):
       header
       aim2
       0 0 0
       0 0 0
   13. Click enter.
       (step IV- converting the file back from aim to isq):
       toisq
       aim2
   14. Copy the isq (file of interest) name: choose views under session manager and click microCT data. Find the file you are working on, and middle click on the file. Then middle click back in the window of the DECterm. Erase (using "backspace") the ".ISQ" at the end of the file, and write:"_new.ISQ" to recognize the aligned file.
   15. Click enter | enter.
   16. Wait until receiving a message: "toisq_from_aim completed"
       (step V- flip):
       isq
       a
   17. Copy the new isq (file of interest) name: choose views under session manager and click microCT data. Find the file being working on, and middle click on the file. Then middle click back in the window of the DECterm.
   18. Click enter | enter. Wait to receive a message: "isq_to_aim completed"
       flip
       a
       aa
       zx
       wait to receive a message: "flip_aim completed"
       from
       aa
   19. Copy the new isq name: choose views under session manager and click microCT data. Find the file being working on, and middle click on the file. Then middle click back in the window of the DECterm. Erase (using "backspace") the ".ISQ" at the end of the file, and write: "_flip.ISQ" to recognize the aligned file.
   20. Wait until receiving a message: "from_aim_to_isq completed"
7. Contouring
   1. Choose the slice which represents approximately the middle of the tooth, in which the coronal pulp, radicular pulp of both canals, and both apical foramens are presented.
   2. Manually mark the contour of interest on the middle slice by clicking on the upper left contour panel- starting from the distal point of the distal root apex mark the apical border coronally to the radiopaque 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 (See Figure 3 II,IV).
   3. Copy this contour (Ctrl+C, Ctrl+V) and adjust it accordingly to 5 slices positioned on either side of the middle slice.
   4. To calculate the tissue volume of the contour marked for each sample choose task under µCT evaluation, and click on 3D evaluation. In the window of 3D evaluation click select near task and choose a filter to calculate the tissue volume (TV).
8. Histology
   1. After the removal of the tissues from the micro-CT scanner, decalcify the tissues by putting each sample in a microcentrifuge tube with 1 mL of ethylenediaminetetraacetic acid (EDTA) 0.5 M pH 8.0 for 10 days. Change the EDTA solution every 3 days.
   2. Dehydration: Put samples in histological cassettes, and into increasing ethanol concentrations, an hour each, as follows: 70% ethanol (at this point the process can be stopped for several weeks, as long as the samples are kept in Ethanol), 90% ethanol 2x, 100% ethanol 2x, xylene 2x (caution: use xylene in a chemical hood, and according to its MSDS guidelines).
      1. Transfer cassettes to liquid paraffin (60 °C) and leave in chemical hood overnight for the xylene to evaporate.
   3. Blocking: Use a histological blocking machine to embed the samples in paraffin. Be careful to embed them in a correct position (i.e., the crown and roots should be oriented parallel to the bottom part of the mold, in a way that the tooth is "lying down") in order to get sagittal sections. The samples are now ready to be cut with a microtome.
   4. Cutting the sections: Start by cutting thick slices of ~20 µm until reaching the relevant part of tissue. Once recognizing any part of the tooth (crown or roots) change to sections of 6 µm, and change the angle of cutting according to the previous section.
      1. For example, if the previous section included the crown but not the roots, change the angle of the block in the microtome so that the roots come closer to the knife, and the crown goes back (the parts of the tooth can be recognized with a naked eye on the block itself).
      2. Continue to adjust the angle until obtaining sagittal sections including coronal and radicular pulp, and the apical foramen. Cut in this orientation as many slices as possible, until the relevant tissue is all cut.
   5. For staining protocols (e.g., Haemotoxylin and Eosin staining (H&E), Brown & Brenn staining, Tartrate-Resistant Acid Phosphatase (TRAP) staining, immunohistochemistry), see^20,21.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Atipamezole hydrochloride	Eurovet Animal Health	CAS 104075-48-1
ATR	dentsply	tecnika
blocking machine	Leica	EG1150H
buprenorphine	vetmarket	163451
clinical microscope/binocular	Olympus	Sz61
dental bur	Komet dental	ZR8801L 315 008
dental spatula	Premier	1003737
EDTA	J.T Baker	8993
entelan	mercury	1.07961
Eosin Y solution, alcoholic	SIGMA	HT110116
hematoxylin solution, Mayer's	SIGMA	MHS 16
Ketamine hydrochloride	Vetoquinol	CAS 1867-669
Medetomidine hydrochloride (cepetor)	CP-pharma GmbH	CAS 86347-15-1
Mepivacaine HCl 3%	Teva	CAS 96-88-8
microbrushes- adjustable precision applicators	PARKELL	S379
micro-ct scanner	scanco	uCT 40
parafin	Leica	39602004
PBS	SIGMA	D8537
PFA	EMS	15710
Chloramphenicol eye ointment (5%)	Rekah pharmaceutical	CAS 56-75-7
tweezers	WAM	Ref-CT
xylazine	Eurovet Animal Health	CAS 7361-61-7
xylene	Gadot	CAS 1330-20-7