Name: induction of periodontitis via a combination of ligature and lipopolysaccharide injection in a rat model
Uploaded: 2023-02-17T14:10:00
Description: Watch this Scientific Journal Video about Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model at JoVE.com

This work was supported by Fundaci&#243; Universitat-Empresa de les Illes Balears (Proof of concept call 2020), by the Instituto de Salud Carlos III, Ministerio de Econom&#237;a y Competividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (contract to M.M.B; FI18/00104) and by the Direcci&#243; General d&#39;Investigaci&#243;, Conselleria d&#39;Investigaci&#243;, Govern Balear (contract to M.M.F.C; FPI/040/2020). The authors thank Dr. Anna Tom&#225;s and Maria Tortosa for their help at the experimental surgery and platform of IdISBa. Finally, thanks to ADEMA School of Dentistry for the access to the CBCT scanner.

NOTE: The experimental protocol of the study was approved by the Ethical Committee of Animal Experimentation of the Balearic Islands Health Research Institute (CEEA-UIB; reference number 163/03/21).
1. Animal anesthesia and procedure preparation
<ol>
	<li>Sterilize all surgical instruments (aluminium mouth gags, dental explorer, diamond lance, surgical scissors, microsurgical pliers, a micro needle holder, a hollenback carver, a periosteal microsurgical elevator, and microsu...

<ol>
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W., Glogauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+ligature-induced+model+of+murine+periodontitis+for+the+evaluation+of+oral+neutrophils.">Robust ligature-induced model of murine periodontitis for the evaluation of oral neutrophils.</a> Journal of Visualized Experiments. 2020 (155), 6-13 (2019).</li><li>Cheng, R., Wu, Z., Li, M., Shao, M., Hu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1&#946;+is+a+potential+therapeutic+target+for+periodontitis:+a+narrative+review.">Interleukin-1&#946; is a potential therapeutic target for periodontitis: a narrative review.</a> International Journal of Oral Science. 12 (1), 1-9 (2020).</li><li>Abe, T., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+ligature-induced+periodontitis+model+in+mice.">Optimization of the ligature-induced periodontitis model in mice.</a> Journal of Immunological Methods. 394 (1-2), 49-54 (2013).</li><li>Jeong-Hyon, K., Bon-Hyuk, G., Sang-Soo, N., Yeon-Cheol, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+rat+models+of+periodontitis+treated+with+natural+extracts.">A review of rat models of periodontitis treated with natural extracts.</a> Journal of Traditional Chinese Medical Sciences. 7 (2), 95-103 (2020).</li><li>Marchesan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+murine+model+to+study+periodontitis.">An experimental murine model to study periodontitis.</a> Nature Protocols. 13 (10), 2247-2267 (2018).</li><li>Lin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+ligature-induced+periodontitis+in+mice+to+explore+the+molecular+mechanism+of+periodontal+disease.">Application of ligature-induced periodontitis in mice to explore the molecular mechanism of periodontal disease.</a> International Journal of Molecular Sciences. 22 (16), 8900 (2021).</li><li>Irie, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+micro-computed+tomography+for+bone+evaluation+in+dentistry.">Use of micro-computed tomography for bone evaluation in dentistry.</a> Brazilian Dental Journal. 29 (3), 227-238 (2018).</li><li>Haas, L. F., Zimmermann, G. 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This method describes the induction of PD in rats following a combined technique of Pg-LPS injections and ligature placement around the M1, revealing that significant changes in the periodontal tissues and alveolar bone could be induced in 14 days following this method.
During this procedure, attention to different critical steps must be provided. During animal anesthesia and procedure preparation, assessing the proper anesthesia during the surgical process is critical for its success...

Periodontitis (PD) is the sixth most prevalent public health condition worldwide, affecting approximately 11% of the total population, being an advanced, irreversible, and destructive form of periodontal disease1,2. PD is an inflammatory process that affects the gingival and periodontal tissues, which results in gingiva recession, apical migration of the junctional epithelium with pocket development, and the loss of alveolar bone3. Furthermore, PD is associated with several systemic diseases, including cardiovascular disease, obesity, diabetes, and rheumatoid arthritis, for which environmental and host-specific factors play a significant role4,5.
Hence, PD is a multifactorial disease primarily initiated by the accumulation of microbial plaque - resulting from dysbiosis of microbial communities - and by an exaggerated host immune response to periodontal pathogens, which leads to the breakdown of periodontal tissue4,6. Among several periodontal bacteria, the gram-negative anaerobic bacterium Porphyromonas gingivalis is one of the key pathogens in PD4. P. gingivalis contains a complex lipopolysaccharide (LPS) in its walls, a molecule known to induce polymorphonuclear leukocyte infiltration and vascular dilatation in inflamed periodontal tissues7. This results in the production of inflammatory mediators, such as interleukin 1 (IL-1), IL-6, and IL-8, tumor necrosis factor (TNF), or prostaglandins, with a subsequent osteoclast activation and bone resorption, leading to tissue destruction and ultimate tooth loss3.
Among the different advantages of animal models include the capacity to mimic cellular complexities as in humans, or to be more accurate than in vitro studies, which are carried out on plastic surfaces with limited cell types8. For modeling PD experimentally in vivo, different animal species have been used, like non-human primates, dogs, pigs, ferrets, rabbits, mice, and rats9. However, rats are the most extensively studied animal model for the pathogenesis of PD because they are inexpensive and easy to handle10. Their dental gingival tissue has similar structural features to human gingival tissue, with a shallow gingival sulcus and junctional epithelium attached to the tooth surface. Furthermore, as in humans, the junctional epithelium facilitates the passage of bacterial, foreign materials, and exudates from inflammatory cells 9.
One of the most reported experimental models of PD induction in rats is the placement of ligatures around the teeth, which is technically challenging but reliable10. The ligature placement facilitates dental plaque and bacterial accumulation, generating a dysbiosis in the gingival sulci, that causes periodontal tissue inflammation and destruction11. Loss of periodontal attachment and resorption of alveolar bone could occur in 7 days in this rat model8.
Another animal model for PD consists of the injection of LPS into the gingival tissue. As a result, osteoclastogenesis and bone loss are stimulated. The histopathological features of this model are similar to human-established PD, characterized by higher levels of proinflammatory cytokines, collagen degradation, and alveolar bone resorption6,8.
Thus, the aim of this study was to describe a simple rat model of experimental PD based on the techniques of P. gingivalis-LPS (Pg-LPS) injections, combined with ligature placement around the first maxillary molars (M1). This is a model with similar characteristics to those observed in human PD disease, which could be used in the study of disease progression mechanisms and future possible treatments.

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			<td>Adsorbent paper point n&#186;30&#160;</td>
			<td>Proclinc</td>
			<td>8187</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma-Aldrich</td>
			<td>A1153</td>
			<td></td>
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		<tr>
			<td>Atipamezole</td>
			<td>Dechra</td>
			<td>573751.5</td>
			<td>Revanzol 5 mg/mL</td>
		</tr>
		<tr>
			<td>Braided silk ligature (5/0)&#160;</td>
			<td>Laboratorio Arago Sl</td>
			<td>613112</td>
			<td></td>
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			<td>Buprenorphine&#160;</td>
			<td>Richter pharma</td>
			<td>578816.6</td>
			<td>Bupaq 0.3 mg/mL</td>
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		<tr>
			<td>Cone-beam computed tomography (CBCT) Scanner&#160;</td>
			<td>MyRay</td>
			<td>hyperion X9</td>
			<td>Model Hyperion X9</td>
		</tr>
		<tr>
			<td>CTAn software</td>
			<td>SkyScan</td>
			<td></td>
			<td>Version 1.13.4.0</td>
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			<td>Dental explorer&#160;</td>
			<td>Proclinc</td>
			<td>99743</td>
			<td></td>
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			<td>Diamond lance-shaped bur&#160;</td>
			<td>Dentaltix</td>
			<td>IT21517</td>
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			<td>Food maintenance diet</td>
			<td>Sodispain research</td>
			<td>ROD14&#160;</td>
			<td></td>
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			<td>Heated surgical platform</td>
			<td>PetSavers</td>
			<td></td>
			<td></td>
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			<td>Hollenback carver</td>
			<td>Hu-FRIEDY&#160;</td>
			<td>HF45234</td>
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			<td>Hypodermic needle&#160;&#160;</td>
			<td>BD&#160;</td>
			<td>300600</td>
			<td>25G X 5/8&#8221; - 0,5 X 16 MM</td>
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			<td>Isoflurane&#160;</td>
			<td>Karizoo</td>
			<td></td>
			<td>Isoflutek 1000mg/g</td>
		</tr>
		<tr>
			<td>Ketamine&#160;&#160;</td>
			<td>Dechra</td>
			<td>581140.6</td>
			<td>Anesketin 100 mg/mL</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide&#160; derived from P.Gingivalis&#160;</td>
			<td>InvivoGen</td>
			<td>TLRL-PGLPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>M/4000/PB08</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holter</td>
			<td>Fehling Surgical Instruments</td>
			<td>KOT-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical pliers</td>
			<td>KLS Martin</td>
			<td>12-384-06-07</td>
			<td></td>
		</tr>
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			<td>microsurgical scissors&#160;</td>
			<td>S&#38;T microsurgical instruments</td>
			<td>SDC-15 RV</td>
			<td></td>
		</tr>
		<tr>
			<td>Monitor iMEC 8 Vet</td>
			<td>Mindray&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiplex bead immunoassay</td>
			<td>Procartaplex, Thermo fisher Scientific</td>
			<td>PPX-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>8187151000</td>
			<td></td>
		</tr>
		<tr>
			<td>Periosteal microsurgical elevator&#160;</td>
			<td>Dentaltix</td>
			<td>CU19112468</td>
			<td></td>
		</tr>
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			<td>Phenylmethylsulfonylfluoride (PMSF)&#160;</td>
			<td>Roche</td>
			<td>10837091001</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution (PBS)</td>
			<td>Capricorn Scientific</td>
			<td>PBS-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP&#160;</td>
			<td>Roche</td>
			<td>4906845001</td>
			<td>Commercial phosphatase inhibitor tablet&#160;</td>
		</tr>
		<tr>
			<td>Plastic vial</td>
			<td>SPL Lifesciencies</td>
			<td>60015</td>
			<td>1.5mL</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Cinfa</td>
			<td>204024.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td>Model SteREO Discovery.V12</td>
		</tr>
		<tr>
			<td>Surgical loupes led light</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors&#160;</td>
			<td>Zepf Surgical</td>
			<td>08-1701-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe&#160;</td>
			<td>BD plastipak</td>
			<td>303172</td>
			<td>1mL</td>
		</tr>
		<tr>
			<td>Veterinary dental micromotor</td>
			<td>Eickemeyer</td>
			<td>174028</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Calier</td>
			<td>20102-003</td>
			<td>Xilagesic 20 mg/mL</td>
		</tr>
	</tbody>
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induction of periodontitis via a combination of ligature and lipopolysaccharide injection in a rat model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1&#946;, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1&#946; levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

A timeline of the experimental steps is presented in Figure 1. Figure 2A shows an image of the mandibula after surgical intervention, with ligature placement around the sulcus of the M1 at time 0 of the experiment. Figure 2B shows how, after 14 days of the procedure, the ligature around the M1 enters the gingival sulcus, causing inflammation of the gingiva and infiltrating accumulation.
<p class="jove_content biglegend" fo:keep-...

Watch this Scientific Journal Video about Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model at JoVE.com

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, ...

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