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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 10  |  Issue : 1  |  Page : 36-40

Methodology in Wistar rats periodontitis induction: A modified ligation technique with injection of bacteria


1 Department of Periodontology, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia
2 Doctor of Dentistry Program, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia

Date of Submission01-Nov-2019
Date of Acceptance11-Feb-2020
Date of Web Publication16-Jul-2020

Correspondence Address:
Prof. Dr. Suryono Suryono
Department of Periodontology, Faculty of Dentistry, Universitas Gadjah Mada, Jalan Denta Sekip Utara, Yogyakarta 55581
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijohs.ijohs_44_19

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  Abstract 


Introduction: Periodontitis is an inflammation affecting the supporting tissue of the teeth. It is characterized by periodontal ligament destruction and progressive alveolar bone loss. Periodontitis is often induced in laboratory animal models, such as Wistar rats used for experimental models. This study was conducted to develop a reproducible technique for induction of periodontitis in the mandible of Wistar rats using a modified ligation technique with an additional injection of Aggregatibacter actinomycetemcomitans.
Context: Periodontitis is an inflammation affecting the supporting tissue of the teeth. It is characterized by periodontal ligament destruction and progressive alveolar bone loss.
Aims: The aim of this study was to develop a reproducible technique for induction of periodontitis in the mandible of Wistar rats using a modified ligation technique with an additional injection of A. actinomycetemcomitans.
Settings and Design: The study was conducted at LPPT Unit IV Universitas Gadjah Mada,
Yogyakarta.
Subjects and Methods: Ten 2-month-old male adult Wistar rats were used as experimental subjects with an average weight of 150–200 g. Nonresorbable silk ligature wire (4/0) was used as ligatures and placed with an “8”-shaped knot technique. The ligatures were placed in between both central incisors of the mandible for 7 days with additional injections of A. actinomycetemcomitans on the 1st day. The procedure was performed under ketamine anesthesia. After 7 days, ligatures were removed and the animal subjects were euthanized with cervical dislocation method and samples of mandibles were preserved in formalin solution and processed histologically and radiographically.
Results: Modified induction technique of periodontitis with ligatures and additional injection of A. actinomycetemcomitans showed significant clinical inflammation, periodontal ligament widening, attachment loss, and alveolar bone loss.
Conclusions: We demonstrated clinical, radiographic, and histological evaluation from this modified induction technique and concluded that it has several advantages: shorter period of induction time with more significant clinical changes and advanced bone loss, simplifying the technique for induction of periodontitis.

Keywords: Aggregatibacter actinomycetemcomitans, ligation, periodontitis induction, Wistar rats


How to cite this article:
Suryono S, Wulandari FR, Andini H, Widjaja J, Nugraheni TD. Methodology in Wistar rats periodontitis induction: A modified ligation technique with injection of bacteria. Int J Oral Health Sci 2020;10:36-40

How to cite this URL:
Suryono S, Wulandari FR, Andini H, Widjaja J, Nugraheni TD. Methodology in Wistar rats periodontitis induction: A modified ligation technique with injection of bacteria. Int J Oral Health Sci [serial online] 2020 [cited 2020 Oct 25];10:36-40. Available from: https://www.ijohsjournal.org/text.asp?2020/10/1/36/289880




  Introduction Top


Periodontitis is a chronic inflammation disease on periodontal tissue which is caused by anaerobe Gram-negative bacteria such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia. Bacteria can trigger periodontal disease directly by releasing endotoxin and indirectly by acting as an immunosuppressive factor.[1] Bacteria and its by-products which spread into the subgingival connective tissue through the junctional epithelium will trigger epithelial cells and fibroblasts to secrete various proinflammatory cytokines. These cytokines can cause connective tissue destruction and alveolar bone resorption in periodontitis.[1],[2]

Animal models which are widely used in periodontal studies are rodents, such as rats. Because rats' gingival structure is quite similar to humans with shallow gingival sulcus and junctional epithelium attached to tooth surfaces. Moreover, the junctional epithelium of both gingivae is a pathway for foreign substances, bacterial toxin, and exudates of inflammatory cells.[3]

Periodontitis model was obtained with bacterial inoculation, high carbohydrate diet, and ligature placement in between teeth. Induction of periodontitis using ligation technique is the most commonly used technique. This technique is performed by twisting cotton, silk, or nylon ligature wire in between the cervical area of maxillary or mandibular molar teeth, resulting in plaque accumulation and epithelial damage. The changes observed in periodontal tissue include destruction and apical migration of the junctional epithelium, accumulation of inflammatory cells, attachment loss, and bone loss. These changes are similar to those happened in humans with periodontitis.[3],[4]

The objective of this study was to develop a reproducible experimental technique for induction of periodontitis in the mandible of Wistar rats using a modified ligation technique with an additional injection of A. actinomycetemcomitans.


  Subjects and Methods Top


The study was conducted at LPPT Unit IV Universitas Gadjah Mada, Yogyakarta. Ten male Wistar rats (Rattus norvegicus) aged 2 months with an average weight of 150–200 g were induced with periodontitis. Animal models were adapted for 3 days before treatment. Wistar rats were fed with AD2 pellets and reverse osmosis water. The induction of periodontitis was performed using a modified ligation technique for 7 days with an additional injection of A. actinomycetemcomitans bacteria on the 1st day. Treatment of experimental animals is carried out from September to October 2018.

General anesthesia was performed through intramuscular injection of ketamine 10% and xylazine 2% (1:1) with 0.5 mL/kg body weight dosage in the lower right thigh muscle 5 min before treatment. Nonresorbable silk ligature wire (4/0) was used as ligatures and placed with an “8”-shaped knot technique in between both mandibular central incisors. Ligature acts as a place for plaque accumulation and causes irritation to the gingiva. Injection of 0.05 mL of A. actinomycetemcomitans 109 CFU/mL was given to the interdental gingiva between both mandibular central incisors. A. actinomycetemcomitans is one of the main causes of localized aggressive periodontitis and commonly identified in chronic periodontitis.[5]

Control of ligatures and animal models was carried out daily. Rats that were sick and died during the treatment were excluded from the study. Ligatures were removed after 7 days, and then, the animal models were euthanized by cervical dislocation using anesthesia with ketamine 10% (1 mL/kg body weight). Samples were stored in formalin solution for radiographic and histological evaluation. This study has obtained a research permission with the number of ethical clearance: no. 001501/KKEP/FKG-UGM/EC/2018.

Analysis method

Radiographic evaluation

Mandible decapitation of the Wistar rats was done using surgical scissors, and then, the samples were cleaned of skin and muscle tissues and then irrigated using NaCl 0.9%. The samples were fixed for 24 h in 10% formalin. Radiographs were taken using a digital periapical radiograph machine PaloDEx Oy (PaloDEx Group, Finland). Conus was positioned upright to the sample, and film distance should be 0.5–1 cm.

Histological evaluation

Mandibular samples were fixed with 10% formalin for 24 h and then decalcified in 8% formic acid solution for 7 days. Dehydration through successive baths of alcohol (70%, 90%, 95%, and 100%) then clarified in xylene. The samples were put into liquid paraffin to form paraffin blocks which will be cut using microtome at 4 μm thickness. Histological preparations were stained using hematoxylin and eosin. The observation of samples was carried out under a Nikon YS 100 Biological Microscope (Nikon Instech, Japan) on ×400 magnification with OptiLab® Viewers (Miconos, Indonesia)”.


  Results Top


The modified ligation technique in this research can be used in induction of periodontitis. The ligatures that were placed 7 days in the mandible with injection of bacteria A. actinomycetemcomitans- induced inflammation of periodontal tissue, as shown in [Figure 1]a. Significant clinical changes can be seen 5 days after induction started. The color of gingiva changed from light pink to bright red. Plaque accumulation can be observed around the ligatures. These changes can be observed within 5 days [Figure 1]b. Clinical changes in [Figure 1]c compared to [Figure 1]a showed macroscopically detectable gingival recession, tooth malposition, and tooth mobility.
Figure 1: Clinical appearances after placement of ligatures and bacterial injection (Aggregatibacter actinomycetemcomitans) on lower central incisors – day 0 (a), day 5 (b) and after day 7 (c)

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Radiographic evaluation

Radiographic evaluation was carried out using digital periapical radiograph in the mandible of periodontitis-induced Wistar rats. As seen in [Figure 2], alveolar ridge resorption was an indicator of bone loss. The reduction of cortical bone density can be detected by decrease in radiopacity. Widening of periodontal ligament can be seen in radiograph as the result of inflammatory process.
Figure 2: Periapical radiographs showing rats' lower jaw in 7 days after induction

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Descriptive histology

Histological evaluation was performed in histological specimen stained with hematoxylin and eosin. Histological aspect in the periodontal tissue that can be observed was attachment loss of the junctional epithelium [Figure 3]a. Granulation tissue can be detected in the interdental gingiva [Figure 3]b. Bone destruction [Figure 3]c can be confirmed with the presence of osteoclast accumulation in the peripheral aspect of the alveolar bone that caused resorption [Figure 4]a. Inflammatory cell infiltration including lymphocyte and macrophage [Figure 4]b can be observed in periodontal ligament as a sign of inflammation.
Figure 3: Histopathological appearance (×100) after induction of periodontitis for 7 days, showing attachment loss (a), granulation tissue (b), and bone destruction (c)

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Figure 4: Histopathological appearance (×400) after induction of periodontitis for 7 days, showing osteoclasts (a) and inflammatory cell infiltration (b) including lymphocyte and macrophage

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  Discussion Top


Periodontitis is one of oral diseases with the second highest prevalence in Indonesia according to Riset Kesehatan Dasar (Indonesia Basic Health Research) 2013. Animal models are widely used by researchers to observe the pathogensis of periodontitis and develop alternative therapies that can be used to support periodontitis healing process.[3],[6],[7],[8]

Ligature causes injury to the sulcus epithelium and increases plaque accumulation that results in easier invasion of A. actinomycetemcomitans. The ligation technique in gnotobiotic rat did not significantly cause gingival inflammation and bone loss. The additional injection of A. actinomycetemcomitans will increase osteoclastogenesis and bone resorption. Periodontitis can be confirmed by the appearance of inflammatory infiltrate in gingiva which will initiate bone resorption. Host response will be reduced due to decreased tolerance to endotoxin produced by bacteria A. actinomycetemcomitans.[9]

Our study proves that ligation technique modified with injection of A. actinomycetemcomitans can accelerate the occurrence of periodontitis in Wistar rats compared to previous studies. Signs and symptoms of periodontitis in our study were significantly observed after 5 days. Studies conducted by Bezerra et al.[10] and Xieet al.[11] showed that induction of periodontitis with ligature technique has significant inflammatory changes after 7 days. Study conducted by Ionel et al.[12] with the same conventional ligature technique showed significant inflammatory changes after 14 days. Literature review conducted by Santana et al.[13] showed that bone loss in periodontal disease induced by ligature technique reaches its peak only after 15 days. The method of injecting A. actinomycetemcomitans suspension to induce periodontitis used by Molon et al.[4] and Garlet et al.[14] in their studies which was carried out for 7 days with repetition in 48 and 96 hours later only showed significant alveolar bone damage after 4 weeks (28 days).

Our study proves that the induction of periodontitis can be accelerated with modification by combining 2 conventional induction techniques, therefore further studies requiring periodontitis induction in animal models can be conducted in lesser period of time. Thus, our study is reproducible for further periodontal studies requiring periodontitis induction in animal models. Animal models also received less trauma compared to conventional repetitive injection technique, requiring only 1 injection on the first day of induction to induce periodontitis in less period of time (7 days compared to 28 days[4],[14]).

This study has some limitations. The research in finding the most effective technique of periodontitis induction has never been done before, so it requires further research. This limitation led to failure in our first experiment by injecting only A. actinomycetemcomitans to the maxillary right molar to induce periodontitis. We made modifications to this method by combining “8”-shaped ligature knot technique with bacterial injection and the occurence of periodontitis was accelerated, but our study was conducted only in mandibular central.


  Conclusions Top


This research explains clinical, radiographic, and histological evaluation of periodontitis induction by a modified ligation technique combined with bacterial injection. This technique has several advantages: short-term induction in 7 days, along with significant clinical changes starting from the 5th day. Clinical evaluation of progressive alveolar resorption was also confirmed.

Financial support and sponsorship

Hibah Dana Masyarakat year 2018 (Faculty Funded).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Newman M, Takei H, Klokkevold P. Carranza's Clinical Periodontology. 12th ed. Missouri: Elsevier; 2015.  Back to cited text no. 1
    
2.
Herbert BA, Novince CM, Kirkwood KL. Aggregatibacter actinomycetemcomitans, a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis. Mol Oral Microbiol 2016;31:207-27.  Back to cited text no. 2
    
3.
Struillou X, Boutigny H, Soueidan A, Layrolle P. Experimental animal models in periodontology: A review. Open Dent J 2010;4:37-47.  Back to cited text no. 3
    
4.
de Molon RS, de Avila ED, Cirelli JA. Host responses induced by different animal models of periodontal disease: A literature review. J Investig Clin Dent 2013;4:211-8.  Back to cited text no. 4
    
5.
Garlet GP, Avila-Campos MJ, Milanezi CM, Ferreira BR, Silva JS. Actinobacillus actinomycetemcomitans-induced periodontal disease in mice: Patterns of cytokine, chemokine, and chemokine receptor expression and leukocyte migration. Microbes Infect 2005;7:738-47.  Back to cited text no. 5
    
6.
Oz HS, Puleo DA. Animal models for periodontal disease. J Biomed Biotechnol 2011;2011:754857.  Back to cited text no. 6
    
7.
Kenworthy R, Baverel M. Studies of a periodontal tissue lesion in the rat, untreated or treated with chlorhexidine digluconate. J Clin Periodontol 1981;8:349-58.  Back to cited text no. 7
    
8.
Rovin S, Costich ER, Gordon HA. The influence of bacteria and irritation in the initiation of periodontal disease in germfree and conventional rats. J Periodontal Res 1966;1:193-203.  Back to cited text no. 8
    
9.
Graves DT, Fine D, Teng YT, Dyke TE, Hajishengallis G. Interactions related to periodontal diseases. J Clin Periodontol 2009;35:89-105.   Back to cited text no. 9
    
10.
Bezerra MM, Brito GA, Ribeiro RA, Rocha FA. Low-dose doxycycline prevents inflammatory bone resorption in rats. Braz J Med Biol Res 2002;35:613-6.  Back to cited text no. 10
    
11.
Xie R, Kuijpers-Jagtman AM, Maltha JC. Inflammatory responses in two commonly used rat models for experimental tooth movement: Comparison with ligature-induced periodontitis. Arch Oral Biol 2011;56:159-67.  Back to cited text no. 11
    
12.
Ionel A, Lucaciu O, Moga M, Buhatel D, Ilea A, Tabaran F, et al. Periodontal disease induced in Wistar rats: Experimental study. HVM Bioflux 2015;7:90-5.  Back to cited text no. 12
    
13.
Santana JB, Moraes RM, Zutin EA, Melo GF, Franco GC, Anbinder AL. The ligature-induced periodontitis model: Literature overview and description of the technique. In: Wallace E, editor. Periodontal Disease: Diagnosis, Management Options and Clinical Features. New York: Nova Biomedical; 2016.   Back to cited text no. 13
    
14.
Garlet GP, Cardoso CR, Silva TA, Ferreira BR, Avila-Campos MJ, Cunha FQ, et al. Cytokine pattern determines the progression of experimental periodontal disease induced by Actinobacillus actinomycetemcomitans through the modulation of MMPs, RANKL, and their physiological inhibitors. Oral Microbiol Immunol 2006;21:12-20.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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