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^5,6,7,8. Historically, it was hypothesized that PD pathogenesis is dependent on large quantities of specific bacteria such as Porphyromonas gingivalis⁹. However, recent evidence suggests that the microbial component of PD is mediated by the dental biofilm. The biofilm is an organized, complex community of numerous microorganisms that can exist in healthy symbiotic and destructive dysbiotic states^10,11. The oral biofilm normally affords resistance to the host by preventing the establishment of foci of pathogenic bacteria and promotes ideal gingival tissue structure and function through regulation of the host immune response^12,13. Perturbations of the equilibrious relationship between commensal organisms within the oral cavity and the host immune system may lead to alterations in tissue homeostasis, resulting in dysbacteriosis and development of the hallmark clinical and radiographic appearances of PD^{5,10,12,13,14}.

Interestingly, the establishment of an oral dysbacteriosis, while required for the initiation of PD, is not sufficient to drive PD in all individuals, eluding toward the ability of the host immune response to subvert the transition of microbiota between symbiotic and dysbiotic states¹⁵. This places a particular spotlight on the means through which PD influences one of the leading characters of the innate immune system, namely the polymorphonuclear granulocyte (PMN), or neutrophil, from local and systemic perspectives^16,17.

In humans, PMNs are recruited from the circulation at a rate of ~2 x 10⁶ cells/h in healthy periodontal connective tissues, where they are the predominating leukocyte population. Here, they are subsequently expelled from the gingival sulcus into the oral cavity as a component of the gingival crevicular fluid. In the presence of PD, neutrophilia manifests within the circulation and oral cavity, where these effector cells possess a hyperinflammatory phenotype that leads to the aforementioned destruction of the periodontium^{17,18,19,20,21,22}. Therefore, understanding the role of PMNs in PD and other systemic inflammatory conditions is of utmost importance.

Although it is widely accepted that chronic diseases are reciprocally linked to PD, the underlying mechanisms have yet to be elucidated, contributing to difficulties in the management of these morbid and potentially fatal systemic conditions. Multiple experimental animal models, each with unique advantages and disadvantages, have been utilized to study the pathophysiology of PD^23,24. Focusing specifically on murine models, there are a variety of protocols through which the study of PD is facilitated; however, they possess several technical and physiologic shortcomings^{25,26,27,28,29,30,31}.

First, the oral gavage mouse model requires numerous oral inoculations of human periodontal pathogens to generate gingival inflammation and bone loss. Additionally, it is generally preceded by a period of antibiotic treatment to subvert the murine commensal oral flora²⁵. This model often requires specialized training to safely perform the oral gavage, uses only a small fraction of periodontal pathogens from the more complex human oral microbiome, and requires several months to establish alveolar bone loss.

In contrast, chemically induced murine models utilize the oral delivery of trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS), agents commonly used in establishing murine models of colitis over a period of several months to induce periodontal bone loss²⁶. Intraoral and extraoral abscess-based models are available, which involve the murine incisors and tissues of the dorsum as well as calvarium, respectively. In the former abscess model, several injections of bacteria are administered, creating multiple gingival abscesses and a dearth of alveolar bone loss, limiting their use in the study of PD. The latter abscess models are significantly more apt to studying bacterial virulence, inflammation, and bone resorption at sites outside of the oral cavity, which eliminates evaluation of the periodontium and oral microbiome^{27,28,29,30,31}.

Using the ligature-induced model of periodontitis, a braided silk suture has commonly been installed circumferentially around the second molar. As an alternative, a single linear segment of suture material can be inserted between the first and second molars^32,33. The goal of the ligature placement is to facilitate bacterial accumulation and generate dysbiosis within the gingival sulci, resulting in periodontal tissue inflammation and destruction of the tissues composing the periodontium. Most notably, this model is capable of producing significantly more alveolar bone loss compared to the more commonly used oral gavage model³⁴. Further complicating the use of the oral gavage model is the natural resistance by several strains of mice (i.e., C57BL/6) to developing alveolar bone loss. This is also problematic, as this strain is the most frequently used in murine-based animal research³⁵.

Existing procedures described by Marchesan et al. and Abe and Hajishengallis were devised to simplify the technical act of placing the ligature^33,36. Unfortunately, the former protocol requires specialized 3D-printed equipment and possess the potential for premature ligature loss, thereby increasing animal use and the costs associated with additional time spent in the operating room. Furthermore, both protocols generate only small regions of the diseased periodontium available for a study.

The advantages that lie with this technique are grounded in the simultaneous study of oral dysbiosis and immunology that govern the periodontium, utilization of low-cost animals with diverse genetic backgrounds, and simple housing and husbandry practices. As such, goals should be to maximize volumes of diseased tissue and, in attempts to practice the principles of reduction in animal research, reduce animal consumption to a level as low as possible. This requires ensuring that all animals are capable of being included in experimental analyses³⁷. However, it should be noted that no matter which animal model of periodontal disease is utilized, there is no single model that encompasses every element of human PD pathophysiology.

This new protocol employs the placement of a ligature around multiple maxillary molar teeth using instrumentation and materials that are found within most laboratories. It allows a sufficient amount of time to easily and confidently install a ligature that is unlikely to avulse prematurely. Finally, as PMNs coordinate destruction of the periodontium in PD, a novel methodology to recover oral neutrophils in a manner analogous to humans is also presented.

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 week-old male C57BL/6 mice, which should be acclimated to their housing facility, using a 0.5" 26 G sterile hypodermic needle and 1 mL syringe according to approved institutional animal care and use committee (IACUC) guidelines.
   NOTE: A combination of ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthetics rapidly and reliably induces the appropriate level of anesthesia for the duration of the procedure.
2. Assess the anesthetic depth prior to and every 15 min during the procedure as evidenced by loss of the pedal reflex.
3. Position the mouse on a heated surgical platform (Figure 1A). Stabilize the maxilla and mandible in the open position using elastic bands and prop the neck with a cotton roll to help maintain the maxilla in a more horizontal orientation (Figure 1B). Cover the body and tail of the animal to mitigate heat loss during the procedure.
4. Position the surgical microscope at the desired magnification and lighting (if not mounted directly to the microscope) over the oral cavity for visualization of the dentition. While the desired magnification is operator-dependent, optimal visualization of the oral cavity is obtained at 16x.
5. Install a sterile 5-0 braided silk suture around the first (M1) and second (M2) maxillary molars within the gingival sulcus using splinter forceps.
   NOTE: Braided silk sutures of a smaller gauge can be used for this purpose to facilitate faster installation and reduce the possibility of iatrogenic soft tissue damage.
   1. 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 (Figure 2A).
   2. Wrap the suture 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 the suture into the gingival sulcus and remove all slack (Figure 2B).
   3. 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 drive the suture into the gingival sulcus and remove all slack (Figure 2C).
      NOTE: If resistance is observed when inserting the suture between M1 and M2 or M2 and M3, the contact may be open slightly using a standard dental explorer.
   4. Tie the ends of the suture with a surgeon's knot and trim the tails as short as possible. Place the knot in the gingival embrasure between M1 and M2 on the palatal side of the maxillary dentition (Figure 2D).
      NOTE: Steps 1.4.1–1.4.4 can be repeated on the contralateral side, if required.
   5. Sterilize the surgical instruments in a hot glass bead sterilizer between each animal subject.
      CAUTION: The tips of the forceps are extremely sharp and can easily cause oral trauma and significant bleeding. Prepare small segments of gauze to remove blood from the oral cavity and apply pressure to actively bleeding wounds.
6. After the ligature installation, remove the mouse from the surgical apparatus, place into a clean cage under a heat lamp and monitor until fully recovered.
7. 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 (12-h light/12-h dark cycle) for 7–11 days.
   NOTE: Mashed chow decreases the forces required for mastication thereby reducing pain associated with feeding, and aids in preventing premature loss of the installed ligature.

2. Sample collection

1. Euthanize mice according to the approved IACUC guidelines.
   NOTE: Individual euthanasia using a CO₂ chamber followed by cervical dislocation is the preferred method for this protocol. This may be altered depending on experiments that require harvesting of additional tissues.
2. Using a pipette, immediately rinse the oral cavity with 100 µL of sterilized 4 °C 1x phosphate-buffered saline (PBS) without calcium and magnesium for 10 s.
3. Repeat step 2.2 2x and place each rinse in a single 15 mL conical polypropylene sterile test tube.
4. Transfer contents to a 50 mL conical polypropylene sterile test tube by running them through a 40 μm nylon mesh filter.
5. Transfer these contents into a new 15 mL conical polypropylene sterile test tube.
   NOTE: The final transfer of the sample to a smaller tube allows for the improved visualization of the cellular pellet in the upcoming steps.
   1. If desired, remove the molar ligature(s) and place into 300 µL of sterilized 1x PBS (4 °C, without calcium and magnesium) in a separate 15 mL conical polypropylene sterile test tube. Agitate gently and remove the suture from the tube. This sample is treated identically to the oral rinse sample from this point forward.
6. Add 33.3 µL of 16% paraformaldehyde (PFA) to facilitate the sample fixation.
7. Vortex the samples immediately and incubate on ice for 15 min.
8. Fill the tube to 15 mL with 1x PBS to dilute PFA, then centrifuge at 1000 x g and 4 °C for 5 min.
9. Aspirate the supernatant and resuspend the pellet in 1 mL of fluorescence-activated cell sorting (FACS) buffer at 4 °C. Count cells on a hemocytometer or automated cell counter.
10. Centrifuge the sample again at 1000 RCF and 4 °C for 5 min.
11. Aspirate the supernatant and resuspend the pellet in an appropriate volume of FACS buffer for a final concentration of 0.5–1.0 x 10⁶ cells/50 μL of FACS buffer.

3. Antibody staining for flow cytometric analysis

NOTE: Label and chill all required FACS tubes prior to use.

1. Add 1 μL of rat serum and 2 μL of anti-mouse IgG antibody to 50 μL of the sample, vortex immediately, and block on ice for 20 min.
2. Add the appropriate antibodies to each sample, vortex immediately, and incubate on ice for 30 min in the dark.
   NOTE: Selection and volumes of antibodies depend on prior optimization pilot experiments tailored to this specific technique.
3. Wash samples with 1 mL of FACS buffer, vortexing briefly and centrifuging for 5 min at 1000 x g and 4 °C. Repeat this step 2x.
4. Resuspend samples in 250 μL of FACS buffer, cover tubes with paraffin film, wrap in aluminum foil, and hold at 4 °C until analysis.
   NOTE: It is ideal to analyze samples within hours of completing the protocol due to the loss of fluorescent signal during long periods of storage. However, samples may be analyzed 2–3 days later if absolutely required.

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