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

NOTE: The experimental protocol of the study was approved by the Ethical Committee of Animal Experimentation of the Balearic Islands Health Research Institute (CEEA-UIB; reference number 163/03/21).

1. Animal anesthesia and procedure preparation

1. Sterilize all surgical instruments (aluminium mouth gags, dental explorer, diamond lance, surgical scissors, microsurgical pliers, a micro needle holder, a hollenback carver, a periosteal microsurgical elevator, and microsurgical scissors) (5 min at 135 °C) before surgery.
2. Prepare all the solutions needed for the procedure in sterile conditions, as described:
   1. Prepare a mix of Ketamine (60 mg/mL) and Xylazine (8 mg/mL) by mixing 1.6 mL of Ketamine with 1 mL of Xylazine diluted in Phosphate Buffer Solution (PBS)/saline. Store the stock at 4 °C.
   2. Dilute Atipamezole to a final concentration of 0.25 mg/mL in PBS/saline. Store the stock at 4 °C.
   3. Dilute Buprenorphine to a final concentration of 0.03 mg/mL in PBS/saline. Store the stock at 4 °C.
   4. Prepare 1 mL of Pg-LPS (1 mg/mL) in sterile saline. Store the stock at -20 °C.
3. For the experiment, use female and male Wistar rats, aged 12 weeks and weighing 210-350 g at the time of surgery. Keep the animals housed in groups under the appropriate environment and constant conditions (20-24 °C, 12 h per day light-dark cycles), with food and standard water, offered ad libitum.
4. To induce anesthesia, weigh the rat and administer the mix of Ketamine/Xylazine at a concentration of 80/10 mg/kg intraperitoneally (IP), by using a 25 G sterile hypodermic needle and 1 mL syringe.
5. After the rat is anesthetized, place the animal on its back on a heated surgical platform; during the procedure, cover the animal's body to prevent heat loss.
   NOTE: Anesthesia depth is assessed by the loss of pedal reflex during the procedure and by monitoring vital signs. If needed, use a small nose cone to maintain anesthesia with 2% Isoflurane in 100% oxygen. Apply sterile ophthalmic ointment to both eyes after anesthesia induction to protect the corneas and prevent drying. 
6. During the procedures, administer 100% oxygen using a small nose cone, and monitor the pulse rate and oxygen saturation by pulse oximetry.
   NOTE: If the oxygen saturation and pulse rate fall below 95% and 190 bpm, respectively, stop the procedure and place the animal in the lateral decubitus position until reaching normal values.
7. Open the rat mouth using an aluminum mouth gag around the incisors (upper and lower), retracting the tongue with it and stabilizing the maxilla and mandible in an open, comfortable working position, enabling access to mandibular molars.
   NOTE: If the animal needs to be placed in lateral decubitus, remove the mouth gag before changing its position to favor recovery. Once the animal is anesthetized, collect gingival crevicular fluid (GCF) before surgery (sample in basal conditions) (day 0).
8. Collect GCF as described by the following steps:
   1. Place the animal on its back on a surgical platform and stabilize the maxilla and mandible in an open position with aluminum mouth gags.
   2. Collect GCF using a total of four (two for each M1) absorbent paper point nº 30 (0.03 cm diameter x 3 cm length), by inserting it into the gingival crevice (space between gingival epithelium and adjacent enamel) around the mesio-palatal of the M1 until slight resistance. Retain the paper point in the same position for a total of 30 s before immediate removal.
   3. After collection, transfer the paper point immediately into a plastic vial, and store at -80 °C until assay performance.

2. Retentive ligature technique and intragingival Pg -LPS injection

NOTE: The ligature model was created (day 0) by placing a sterile braided silk ligature (5/0) around the M1 bilaterally within the gingival sulcus using microsurgical instruments and securing it with the surgeon's knots on the palatal surface. The microsurgical instruments used were microsurgical pliers, a micro needle holder, a hollenback carver, a periosteal microsurgical elevator, and microsurgical scissors. Surgical loupes with an LED light source were also used (3.6x magnification).

1. Position the distal tail of the suture on the palatal side of the dentition and insert the proximal segment between the contact of M1 and second maxillary molars (M2).
2. Use the periosteal microsurgical elevator to insert the suture within the sulcus. Wrap the ligature around the buccal surface of the M1 very carefully, as the tissues at this level present a narrow zone of attached gingiva. On the palatal aspect, make sure the suture is tightened on both ends to ensure it is driven into the gingival sulcus.
   NOTE: If resistance is observed when inserting the suture between the M1 and the M2, the contact may be opened slightly using a dental explorer and diamond lance-shaped bur.
3. Tie the ends of the suture with a surgeon's knot and trim the tails as short as possible. Insert the knot in the sulcus.
   NOTE: Tips of surgical instruments can cause oral trauma and bleeding. Prepare small segments of gauze or a cotton swab to remove blood from the oral cavity and apply pressure to stop any hemorrhage. Careful soft tissue management with a microsurgical approach minimizes surgical complications and leads to less tissue trauma.
4. After ligature positioning, inject 40 µL of Pg-LPS in sterile saline with a 25 G sterile hypodermic needle and a 1 mL syringe to the subgingival tissue (between the root or neck of a tooth and the gum margin) at the mesio-palatal side of the M1 bilaterally (day 0).

3. End of the procedure

1. After ligation positioning and Pg-LPS application, release the rat from the surgical state and place it in a clean individual cage under a heat lamp.
2. Inject the antagonist Atipamezole (0.5 mg/kg subcutaneously (SC)) with a 25 G sterile hypodermic needle and a 1 mL syringe.
3. For pain relief, inject 0.03 mg/kg Buprenorphine, SC.
4. Monitor the animal's recovery until the effects of the operation are completely reversed. Individually house each rat in the appropriate environment under constant conditions (20-24 °C, 12 h per day light-dark cycles). Offer food and deionized water ad libitum.
5. During the first 2 days after the procedure, weigh the animals and inject Buprenorphine SC twice daily for pain relief.
   NOTE: Buprenorphine can be administered before beginning the procedure to eliminate the windup effect.
6. During the time of the experiment, monitor the animals by weight and general behavior assessment one or two times a week.

4. Post-procedure follow-up

NOTE: The induction of PD was maintained for 14 days to promote the accumulation of bacteria biofilm and consequent inflammation. The ligatures have to be examined and adjusted, and Pg-LPS is injected three times per week (day 2, day 4, day 6, day 8, day 10, and day 12).

1. Examine and adjust the ligature (day 2, day 4, day 6, day 8, day 10, and day 12) as follows:
   1. Anesthetize with 2% isoflurane in 100% oxygen using an anesthesia induction chamber.
   2. After the rat is anesthetized, place the animal on its back and use a small nose cone during the procedure with 1% isoflurane in 100% oxygen for maintenance of the animal anesthesia.
   3. Open the rat mouth using the aluminum mouth gag around the incisors (upper and lower), retracting the tongue with it and stabilizing the maxilla and mandible in an open, comfortable working position, enabling access to ligatures.
   4. Tighten the ligatures against the gingiva with the help of a periosteal microsurgical elevator, and make sure the suture of the ligatures is inserted, creating inflammation around the gingiva.
      NOTE: It is possible that 7-10 days after surgery, ligatures are lost. If this happens, follow the protocol as explained for the injection of Pg-LPS (step 2.4).
2. After the ligature adjustment, bilaterally inject 40 µL of Pg-LPS with a 25 G sterile hypodermic needle and a 1 mL syringe to the subgingival tissue at the mesio-palatal side of the M1 (day 2, day 4, day 6, day 8, day 10, and day 12).
3. Remove the nose cone for anesthesia and place the rat in its cage. Monitor the animal's recovery until the effects of the anesthesia are completely reversed.

5. Animal sacrifice and analysis

NOTE: There are different options for evaluating the progression of PD. Here, the analysis described consists of an evaluation of pro-inflammatory cytokines at the gingival crevicular fluid (GCF), and an evaluation of the loss of alveolar bone.

1. On day 14 of the study (day 14), sacrifice the animals with CO₂ in a carbon dioxide chamber. A displacement rate of 30% to 70% of the chamber volume/min is recommended for rodents.
   NOTE: Lack of animal response to pedal reflex and absence of vital signs must be verified to confirm euthanasia. 
2. Collect GCF as described by the following steps:
   NOTE: GCF is collected before PD induction (before surgery) (day 0) and after PD induction (after sacrifice) (day 14).
   1. Place the animal on its back on a surgical platform, and stabilize the maxilla and mandible in an open position with aluminum mouth gags.
   2. Collect the GCF using adsorbent paper point nº 30 (0.03 cm diameter x 3 cm length) by inserting it into the gingival crevice around the mesio-palatal of the M1 until slight resistance. Retain the paper point in the same position for a total of 30 s before immediate removal.
   3. After collection, transfer the paper point immediately into a plastic vial and store at -80 °C until assay performance.
3. To evaluate proteins within the GCF, prepare the following solutions and follow the steps of the elution method as described:
   NOTE: IL-1β is evaluated on the GCF (day 14) using an immunoassay, according to the manufacturer's protocol.
   1. Prepare the elution buffer fresh and keep it on ice throughout the entire extraction process to inhibit protease activity.
   2. Prepare all solutions needed for the elution buffer as described:
      1. Prepare 1 mL of Aprotinin (1 mg/mL) in ultrapure water.
      2. Prepare 10 mL of phenylmethylsulfonylfluoride (PMSF) (200 mM) in methanol.
      3. Add 125 µL of PMSF and 250 µL of Aprotinin to 24.5 mL of PBS solution (pH = 7.4) to prepare the elution buffer.
   3. Dissolve one commercial phosphatase inhibitor tablet with 10 mL of freshly prepared elution buffer for 10 min under agitation at 4 °C.
      NOTE: The duration of GCF elution is limited; use immediately for centrifugations after the addition of phosphatase inhibitor tablet within the first 30 min.
   4. After adding the phosphatase inhibitor, add 11 µL of complete elution buffer directly onto the tube with the paper points.
   5. Centrifuge the tube at 452 x g for 5 min at 4 °C.
   6. Transfer the eluted content to a new plastic vial. Repeat this process four additional times to yield a 50 µL total volume.
   7. After the last centrifugation, add a total volume of 60 µL directly onto the paper point, and centrifuge one last time at 452 x g for 5 min at 4 °C.
   8. Use the required volume eluted for protein evaluation.
4. After euthanasia and GCF collection, with the help of surgical scissors, cut out the superior jaw of the rats. Try to peel off the mask of the rat and leave as little soft tissue as possible.
5. Position the jaw in the microscope stage for visualization and take a picture at the desired magnification.
6. Place the jaw directly in 4% paraformaldehyde (PFA) diluted in PBS. Refresh twice a week with new PFA for 12 days for a complete fixation.
   CAUTION: Steps involving PFA should be performed in a fume hood following the Safety Data Sheet recommendations.
7. After complete jaw fixation, evaluate bone loss using a cone-beam computed tomography (CBCT) scanner, as follows:
   1. Open the PC.
   2. Open the CBCT analysis program.
   3. Select Scan Protocol > Denture Scan Mode.
   4. Select Field of View (FOV) > (11×8) HIRes (90 kV of voltage and 3 mA of current).
   5. Place the fixed upper maxilla of the rats in the gantry.
   6. Click Next.
   7. Click the X-ray firing button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the scan).
   8. If necessary, relocate the upper maxilla in the center of the frontal plane by pressing the control button, then click the X-ray firing button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the scan).
   9. Click Next.
   10. Click the X-ray firing button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the scan).
   11. If necessary, relocate the denture in the center of the sagittal plane by pressing the control button, then click the X-ray firing button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the scan).
   12. Click Next.
   13. Click the Start button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the exam). Wait until receiving a processing message, and then follow the instructions shown.
   14. A window view appears. Regulate the gray at 65% and click Apply.
   15. For saving the scan, click on File and save it in DICOM format. Then, click All Images and at the DICOM export selection window, select all the parameters shown (initial image, original axial, reformatted axial, multiplanar) and select predefined type.
8. Process the maxillary molars' two-dimensional and sagittal representative images using an analysis program, as follows:
   1. Open the software.
   2. Click File and Open, and then select the folder with images in DICOM format and click Open.
   3. Wait until the window "Convert to 8 bit" appears, select the range from 0 to 6,000, and click OK.
   4. Click Raw images.
   5. Define the top and bottom of the selection to limit the region of interest. Search for the first and last images where alveolar bone appears and select it with the top of selection and bottom of selection commands.
      NOTE: Representative two-dimensional images of the alveolar bone of the molars could be exported, as in Figure 3A,B.
   6. For sagittal representative images, click on Toggle profile bar.
   7. Draw a sagittal line in the middle of the palate and click Reslice model. Determine parameters for delimiting the region of interest (slice spacing: 1; number of slices: 100) and click OK. Wait until the reslicing model ends. Finally, click save Resliced images.
      NOTE: Representative sagittal imagines of the alveolar bone of the molars could be exported, as in Figure 3C,D.