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This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.
The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.
Periodontal disease (PD) is an osteolytic condition associated with significant host morbidity and economic burden, which is manifested by gingival inflammation and loss of both soft tissue attachment and osseous support for the affected dentition1,2,3,4. This process is governed by interactions between the oral microbiota and innate immune system of the host. It is also associated with exacerbation of other systemic inflammatory diseases including diabetes, cardiovascular disease, and cancer5,
All murine studies complied with the relevant ethical regulations and were approved by the University of Toronto Animal Care Committee and the Research Ethics Board (Protocol 20011930).
1. Ligature installation
NOTE: This is a non-sterile surgical procedure that can be carried out in a standard operating theater. The use of germ-free animals (not covered here) mandates handling within a biosafety cabinet, use of sterile instruments, and inoculation of the oral cavity .......
Representative flow cytometry data from oral rinse samples of a naive (Figure 3A) and inflamed (Figure 3B) murine oral cavity secondary to the ligature-induced periodontitis are provided. Recovery of PMNs from an installed ligature is also demonstrated (Figure 3C). Flow cytometer channel voltages were calibrated manually, and compensation was performed with single-stained compensati.......
The most critical element associated with use of the murine ligature-induced model of periodontitis is centered around the retention of the ligature until the time of sacrifice or intentional removal. The installed biofilm-retentive ligature is capable of inducing a significant loss of alveolar bone height in as few as 6 days, plateauing between the 11–16 day period39. The decision to sacrifice animal subjects before the maximal period of bone loss, rendering this a much shorter model of lig.......
J. W. C. is supported by the Canadian Institutes of Health Research (CIHR). The authors would like to thank Dr. Chunxiang Sun for her assistance in performing the trypan blue staining.
....Name | Company | Catalog Number | Comments |
Anti-mouse F4/80 Antibody | BioLegend | 123131 | BV421, Clone BM8 |
Anti-mouse Ly6G Antibody | BD | 560602 | PerCP-Cy5.5, Clone 1A8 |
C57BL/6 Male Mice | Charles River | 8 to 12 weeks old | |
Conical Centrifuge Tube | FroggaBio | TB15-500 | 15 mL |
Conical Centrifuge Tube | FroggaBio | TB50-500 | 50 mL |
FACS Buffer | Multiple | 1% BSA (BioShop), 2mM EDTA (Merck), 1x HBSS-/- (Gibco) | |
FACSDiva | BD | v8.0.1 | |
Fibre-Lite | Dolan-Jenner | Model 180 | |
FlowJo | Tree Star | v10.0.8r1 | |
Heat Therapy Pump | Hallowell | HTP-1500 | |
Hot Glass Bead Sterilizer | Electron Microscopy Sciences | 66118-10 | Germinator 500 |
Iris Scissors | Almedic | 7602-A8-684 | Straight |
Ketamine | Vetoquinol | 100mg/mL | |
LSRFortessa | BD | X-20 | |
Mouse Serum | Sigma | M5905-5ML | |
Nylon Mesh Filter | Fisher Scientific | 22-363-547 | 40 µm |
Paraformaldehyde | Fisher Scientific | 28908 | 16% (w/v), Methanol Free |
Phosphate-buffered Saline | Sigma | D1408-500ML | Without CaCl2 and MgCl2, 10x |
Plastic Disposable Syringes | BD | 309659 | 1 mL |
Rat Serum | Sigma | R9759-5ML | |
Silk Suture | Covidien | SS652 | C13 USP 5-0 |
Splinter Forceps | Almedic | 7726-A10-700 | #1 |
Splinter Forceps | Almedic | 7727-A10-704 | #5 |
Stereo Dissecting Microscope | Carl Zeiss | 28865 | Photo-Zusatz |
Sterile Hypodemic Needle | BD | 305111 | 26G X 1/2" |
Syringe | BD | 309659 | 1 mL |
Xylazine | Rompun | 20mg/mL |
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