This is a rat model of experimental periodontitis, based on the techniques of lipopolysaccharide injections combined with ligature placement, offering an effective method to study disease progression and to evaluate treatments for periodontitis. Combining both techniques with individual methods culminates in a rapid disease induction, amplifying destructive inflammatory response and increasing loss of connective tissue and alveolar bone resorption. This method provides a valuable opportunity to assess the impact of periodontitis treatments.
Demonstrating the procedure will be Dr.Oscar Villa, PhD. Begin by sterilizing all surgical instruments before surgery. Prepare a mix of ketamine and xylazine by mixing 1.6 milliliters of ketamine with one milliliter of xylazine, diluted in PBS or saline, and store the stock at four degrees Celsius.
Dilute atipamezole to a final concentration of 0.25 milligrams per milliliter and buprenorphine to a final concentration of 0.03 milligrams per milliliter in PBS or saline, and store the stocks at four degrees Celsius. Prepare one milliliter of PG-LPS in sterile saline and store the stock at minus 20 degrees Celsius. Use female and male Wistar rats for the experiment.
Keep the animals housed in groups under the appropriate environment, with food and standard water offered ad libitum. After the rat is anesthetized, place the animal on its back on a heated surgical platform. Cover the animal's body during the procedure to prevent heat loss.
Administer 100%oxygen using a small nose cone, and monitor the pulse rate and oxygen saturation by pulse oximetry. Now, open the rat mouth using an aluminum mouth gag around the incisors. Retract the tongue with it and stabilize the maxilla and mandible in an open, comfortable working position, enabling access to mandibular molars.
Collect GCF using a total of four absorbent paper point number 30, by inserting it into the gingival crevice around the mesiopalatal of the M1 until slight resistance. Retain the paper point in the same position for 30 seconds before immediate removal. After collection, transfer the paper point immediately into a plastic vial and store at minus 80 degrees Celsius until assay performance.
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. 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.
Ensure the suture is tightened on both ends to be driven into the gingival sulcus. Next, 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.
After ligature positioning, inject 40 microliters of PG-LPS in sterile saline into the subgingival tissue at the mesiopalatal side of the M1 bilaterally. To examine and adjust the ligature, place the anesthetized animal on its back and use a small nose cone during the procedure with 1%isoflurane and 100%oxygen for maintenance of the animal anesthesia. Open the wrap mouth using the aluminum mouth gag around the incisors, retracting the tongue with it and stabilizing the maxilla and mandible in an open, comfortable working position, enabling access to ligatures.
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. After the ligature adjustment, bilaterally inject 40 microliters of PG-LPS into the subgingival tissue at the mesiopalatal side of the M1, as demonstrated earlier. After sacrificing the animal on the 14th day, collect GCF as shown earlier.
Transfer the paper point immediately into a plastic vial and store at minus 80 degrees Celsius until assay performance. The bi-dimensional images of the alveolar bone of maxillary molars show more bone volume in the basal control and higher alveolar bone loss after establishing periodontitis. Analysis of the sagittal images highlights a more significant alveolar bone loss in the interradicular bone area of the M1 and M2 after periodontitis establishment, compared to the basal control group.
After sacrifice, the palate presented differences in gingival recession in the M1 among different groups. The periodontitis group shows a more remarkable apical gingival migration and inflammation of the gingiva around the maxillary molars compared to the basal control group. Also, the periodontitis group showed a significantly higher release of pro-inflammatory cytokine 1L-1 beta from GCF than the control group.
Crucial steps in the procedure are the correct placement of the suture and knot in the sulcus, proper adjustment of the ligature, and accurate lipopolysaccharide injection during the 14-day protocol.
