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.
