The protocol described here furnishes investigators with a method to study oral neutrophils within a ligature-induced animal model of periodontal disease in an analogous manner to human participants. The main advantage of this technique is highlighted by the increased volume of inflamed gingival tissues available for subsequent analysis, which reduces animal usage. Our ability to retrieve the biofilm-coated ligatures from around the teeth allows for the simulation of effective treatment of a localized periodontal disease.
The use of this model could be applied in the study of the systemic effects of periodontitis in an animal model, due to the increased areas of inflamed gingival tissues. Familiarization with the use of microsurgical instruments and dissection microscopes, as well as tying surgeon's knots prior to handling live animal subjects, is heavily advised. Visual demonstration of this technique is critical in reducing the length of the learning curve required to achieve proficient ligature placement.
Demonstrating the procedure will be Jeff Chadwig, a graduate student from my laboratory. Start by positioning the mouse on a heated surgical platform. After ensuring that the mouse is properly anesthetized, stabilize the maxilla and mandible in the open position using elastic bands.
Prop the neck with a cotton roll to maintain the maxilla in a horizontal position and cover the body and tail to mitigate heat loss. Position the surgical microscope at the desired magnification and lighting over the oral cavity. Use splinter forceps to install a sterile 50 or smaller braided silk suture around the M1 and M2 maxillary molars within the gingival sulcus.
Position the distal tail of the suture on the palatal side of the dentition and insert the proximal segment between the contact of M2 and M3.Then, wrap it around the buccal surface of M2 and insert it between the contact of M1 and M2.Ensure that both ends of the suture are pulled tightly to drive it in to the gingival sulcus. Wrap the proximal suture segment around M1 below its height of contour and insert it between the contact of M1 and M2.Pull the proximal tail of the suture tightly to remove all slack. Tie the ends of the suture with a surgeon's knot and trim the tails as sort as possible.
Place the knot in the gingival embrasure between M1 and M2 on the palatal side of the maxillary dentition. It is critical that the knot used to secure the ligature is secure and in position where it will not irritate the animal or interfere with mastication. After the ligature installation, remove the mouse from the surgical apparatus and place it into a clean cage under a heat lamp, making sure to monitor it until fully recovered.
Sterilize the surgical instruments in the hot glass bead sterilizer between each animal subject. Individually house each mouse with the appropriate environmental enrichment and allow ad libitum access to filtered water and mashed standard chow in a temperature and humidity controlled environment for seven to 11 days. When ready to collect samples, use a pipette to rinse the oral cavity with 100 microliters of sterilized four degree Celsius PBS without calcium and magnesium for 10 seconds.
Repeat the rinse two more times, placing each rinse into the same 15 milliliter conical sterile test tube for each animal. Run the contents of each 15 milliliter tube through a 40 micrometer nylon mesh filter into a 50 milliliter conical tube, and transfer the filtrate into a new 15 milliliter test tube. Add 33.3 microliters of 16%paraformaldehyde to facilitate sample fixation.
Vortex the samples immediately and incubate them on ice for 15 minutes. Paraformaldehyde is the most hazardous reagent employed in the protocol and should be handled with the recommended personal protective equipment advised by the manufacturer. After the incubation, fill the tube to 15 milliliters with PBS then centrifuge it at 1000xg and four degrees Celsius for five minutes.
Aspirate the supernatant, re-suspend the pellet in one milliliter of FACS buffer at four degrees Celsius and count the cells on a hemocytometer or automated cell counter. Repeat the centrifugation and aspirate the supernatant. Then, re-suspend the cell pellet in an appropriate volume of FACS buffer for a final concentration of 500, 000 to 1 million cells per 50 microliters of FACS buffer and transfer the cells to FACS tube.
Add one microliter of rat serum and two microliters of anti-mouse IGG antibody to 50 microliters of the sample. Vortex the sample immediately and block it on ice for 20 minutes. Then, add the appropriate antibodies to each sample.
Vortex and incubate on ice for 30 minutes in the dark. After the incubation, wash the samples with one milliliter of FACS buffer, vortex briefly, and centrifuge for five minutes at 1000xg and four degrees Celsius. Dump the supernatant and gently block the opening of the FACS tube.
Repeat the wash twice, then re-suspend the samples in 250 microliters of FACS buffer for storage. Cover the tubes with paraffin film, wrap them in aluminum foil, and store them at four degrees Celsius until ready to analyze. Flow cytometry was used to analyze neutrophils recovered from an oral rinse from a naive mouse, as well as an oral rinse and recovered ligature from a mouse with ligature-induced periodontitis.
To assess alveolar bone loss, alveolar bone levels were measured from the alveolar bone crest to the cementoenamel junction for healthy and ligated mice. Then, the measurement differences were calculated. While not reviewed here, histopathologic, immunohistochemical, and morphometric evaluation of the periodontium and alveolar bone can also be undertaken depending on the research question.
This model is currently being used in my laboratory to study the effects of the oral innate immune system on systemic health and disease.
