Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

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 dentition^1,2,3,4. 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 cancer^5,

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 with periodontal pathogens to cause the clinical manifestations of periodontitis.

1. Administer intraperitoneal anesthesia to 8–12 we....

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.......

Materials

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