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.

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 ...

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&#8211;16 day period39. The decision to sacrifice animal subjects before the maximal period of bone loss, rendering this a much shorter model of lig...

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 cancer5,6,7,8. 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 states10,11. 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 response12,13. 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 PD5,10,12,13,14.
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 perspectives16,17.
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 periodontium17,18,19,20,21,22. 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 PD23,24. 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 shortcomings25,26,27,28,29,30,31.
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 microbiome27,28,29,30,31.
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 molars32,33. 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 ligature33,36. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-mouse F4/80 Antibody</td>
			<td>BioLegend</td>
			<td>123131</td>
			<td>BV421, Clone BM8</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6G Antibody</td>
			<td>BD</td>
			<td>560602</td>
			<td>PerCP-Cy5.5, Clone 1A8</td>
		</tr>
		<tr>
			<td>C57BL/6 Male Mice</td>
			<td>Charles River</td>
			<td></td>
			<td>8 to 12 weeks old</td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tube</td>
			<td>FroggaBio</td>
			<td>TB15-500</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tube</td>
			<td>FroggaBio</td>
			<td>TB50-500</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>FACS Buffer</td>
			<td>Multiple</td>
			<td></td>
			<td>1% BSA (BioShop), 2mM EDTA (Merck), 1x HBSS-/- (Gibco)</td>
		</tr>
		<tr>
			<td>FACSDiva</td>
			<td>BD</td>
			<td></td>
			<td>v8.0.1</td>
		</tr>
		<tr>
			<td>Fibre-Lite</td>
			<td>Dolan-Jenner</td>
			<td></td>
			<td>Model 180</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>Tree Star</td>
			<td></td>
			<td>v10.0.8r1</td>
		</tr>
		<tr>
			<td>Heat Therapy Pump</td>
			<td>Hallowell</td>
			<td></td>
			<td>HTP-1500</td>
		</tr>
		<tr>
			<td>Hot Glass Bead Sterilizer</td>
			<td>Electron Microscopy Sciences</td>
			<td>66118-10</td>
			<td>Germinator 500</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Almedic</td>
			<td>7602-A8-684</td>
			<td>Straight</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vetoquinol</td>
			<td></td>
			<td>100mg/mL</td>
		</tr>
		<tr>
			<td>LSRFortessa</td>
			<td>BD</td>
			<td></td>
			<td>X-20</td>
		</tr>
		<tr>
			<td>Mouse Serum</td>
			<td>Sigma</td>
			<td>M5905-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Filter</td>
			<td>Fisher Scientific</td>
			<td>22-363-547</td>
			<td>40 &#181;m</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>28908</td>
			<td>16% (w/v), Methanol Free</td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline</td>
			<td>Sigma</td>
			<td>D1408-500ML</td>
			<td>Without CaCl2 and MgCl2, 10x</td>
		</tr>
		<tr>
			<td>Plastic Disposable Syringes</td>
			<td>BD</td>
			<td>309659</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Rat Serum</td>
			<td>Sigma</td>
			<td>R9759-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk Suture</td>
			<td>Covidien</td>
			<td>SS652</td>
			<td>C13 USP 5-0</td>
		</tr>
		<tr>
			<td>Splinter Forceps</td>
			<td>Almedic</td>
			<td>7726-A10-700</td>
			<td>#1</td>
		</tr>
		<tr>
			<td>Splinter Forceps</td>
			<td>Almedic</td>
			<td>7727-A10-704</td>
			<td>#5</td>
		</tr>
		<tr>
			<td>Stereo Dissecting Microscope</td>
			<td>Carl Zeiss</td>
			<td>28865</td>
			<td>Photo-Zusatz</td>
		</tr>
		<tr>
			<td>Sterile Hypodemic Needle</td>
			<td>BD</td>
			<td>305111</td>
			<td>26G X 1/2&#34;</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Rompun</td>
			<td></td>
			<td>20mg/mL</td>
		</tr>
	</tbody>
</table>

robust ligature-induced model of murine periodontitis for the evaluation of oral neutrophils

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.

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...

Watch this Scientific Journal Video about Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils at JoVE.com

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

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 ...

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.

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

Abstract