Murine Model of Advanced Periodontitis Induced by Nylon Ligature in the Second Upper Molar

The scope of our research is to study the cellular and molecular factors that favor periodontitis for which we use a murine model of ligature-induced periodontitis.

Recent research has established the importance of inflammation. Dysbiosis and systemic diseases are determining factors in the development of periodontitis.

The technology recently used in periodontics includes animal models, some look out for different molecules, proteomics sequencing, flow cytometry, and histology, among others.

Current experimental challenges are related to understanding the interaction between systemic diseases and periodontitis.

We use a murine model of ligature periodontitis to demonstrate that the pro-inflammatory cytokine A and S is involved in the development of periodontitis during pregnancy.

[Narrator] To begin, place an anesthetized mouse on a work table facing upward with its head directed toward the operator. Gently pull each leg without tension and secure them with micropore tape to prevent sudden involuntary movements. Cover the mouse with a blanket or gauze to maintain its body heat. Place one end of an orthodontic elastic around the upper incisors and secure the other end to an upper holder without applying tension. Then place a second orthodontic elastic around the inferior incisors and secure it to an inferior holder using a rubber band. Now insert the cheek separators and carefully move the tongue to one side to improve visibility. Position the microscope to visualize the upper molars using the lowest magnification objective. Once located, increased magnification to optimize comfort and focus for the operator. When the image is clear, identify the largest and proximal molar M1, the next molar M2, and the smallest and distal molar M3. Use a 6-0 nylon suture for ligature placement as it causes less mechanical damage compared to wider sutures. Using tissue forceps, hold the distal tip of the 6-0 nylon suture and place it at the base of the papilla from the palatine side at the distal of M2. Apply light pressure through the base of the interproximal palatal space to advance it toward the buccal surface. When the suture crosses, pull it through the interproximal space toward the buccal side. Now, place the tip of the suture at the base of the interproximal area at the mesial surface of M2 from the buccal side. Push it gently to cross the interproximal space and pass it back through the buccal space to the palatine. Gently pull on the 6-0 nylon suture. Hold the tip with tissue forceps adjusted around M2, and secure the suture using three simple knots. Then cut the suture with fine scissors. To release the mouse, first, remove the elastic around the lower incisors and then the cheek separators. Then remove the elastic around the upper incisors and the micropore tape from the legs. Now remove the mouse from the work table and keep the tongue to one side to maintain an open airway and prevent blockage. Wrap the mouse in gauze or cloth and place it face up. Keep the animal warm, covered with cloth or gauze, and under observation until it is completely awake. Apply one drop of hypromellose in each eye until the mouse can blink normally. Then, return it to its regular cage. To check the permanence of the ligature, take the mouse. hold its head and body firmly, and use tissue forceps to open the mouth. Under lamp light, observe the 6-0 nylon suture around M2. For the confirmation of biofilm formation using histology, harvest the maxilli from euthanized animals after 30 days. Wash the tissues in 0.9% sodium chloride solution and place them into labeled microcentrifuge tubes. Fix the samples in 4% paraformaldehyde solution for two hours under agitation. Then, wash the fixed samples with tap water for two hours. Next, decalcify the samples in 20 volumes of 4% EDTA at pH 7.3 for 20 days, changing the EDTA every four days. Then, embed the samples in paraffin. Cut five micrometer sections from the M2 area and stain them with hematoxylin and eosin. Observe the stained sections under an optical microscope to describe changes in sulcus epithelium, gingival and periodontal fibers, height, and alveolar crest integrity. Measure attachment loss by determining the distance between the cemento enamel junction and the highest point of the bone crest using a digital editing program. After histometric analysis, analyze data from control and periodontal treatment groups using the Mann-Whitney U test. Periodontitis induces tissue loss in mice, inflammation, bleeding, and periodontal pocket formation at the gingival margin by day 30, in contrast to the control group. Histological analysis confirmed significant structural damage in the PT group, with presence of periodontal pockets, detachment of fibers, and apical migration of the epithelium. Loss of cell nuclei in gingiva and bone of the buccal and palatal sides were also seen. In contrast, the CTL group showed healthy histology. Quantitative analysis revealed significantly increased attachment loss in the PT group with buccal side loss at approximately 209 micrometers and palatine side at approximately 254 micrometers.