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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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,

Protocol

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 Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Anti-mouse F4/80 AntibodyBioLegend123131BV421, Clone BM8
Anti-mouse Ly6G AntibodyBD560602PerCP-Cy5.5, Clone 1A8
C57BL/6 Male MiceCharles River8 to 12 weeks old
Conical Centrifuge TubeFroggaBioTB15-50015 mL
Conical Centrifuge TubeFroggaBioTB50-50050 mL
FACS BufferMultiple1% BSA (BioShop), 2mM EDTA (Merck), 1x HBSS-/- (Gibco)
FACSDivaBDv8.0.1
Fibre-LiteDolan-JennerModel 180
FlowJoTree Starv10.0.8r1
Heat Therapy PumpHallowellHTP-1500
Hot Glass Bead SterilizerElectron Microscopy Sciences66118-10Germinator 500
Iris ScissorsAlmedic7602-A8-684Straight
KetamineVetoquinol100mg/mL
LSRFortessaBDX-20
Mouse SerumSigmaM5905-5ML
Nylon Mesh FilterFisher Scientific22-363-54740 µm
ParaformaldehydeFisher Scientific2890816% (w/v), Methanol Free
Phosphate-buffered SalineSigmaD1408-500MLWithout CaCl2 and MgCl2, 10x
Plastic Disposable SyringesBD3096591 mL
Rat SerumSigmaR9759-5ML
Silk SutureCovidienSS652C13 USP 5-0
Splinter ForcepsAlmedic7726-A10-700#1
Splinter ForcepsAlmedic7727-A10-704#5
Stereo Dissecting MicroscopeCarl Zeiss28865Photo-Zusatz
Sterile Hypodemic NeedleBD30511126G X 1/2"
SyringeBD3096591 mL
XylazineRompun20mg/mL

References

  1. Hajishengallis, G. Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response. Trends in Immunology. 35 (1), 3-11 (2014).
  2. Pihlstrom, B. L., Michalowicz, B. S., Johnson, N. W. Periodontal diseases.

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