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A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation
JIM-12279; No of Pages 6 Journal of Immunological Methods xxx (2017) xxx–xxx
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A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation Ai Okanobu a, Shinji Matsuda a,⁎, Mikihito Kajiya a, Tsuyoshi Fujita a, Mizuho Kittaka a, Hideki Shiba b, Hidemi Kurihara a a b
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Japan Department of Biological Endodontics, Integrated Health Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Japan
a r t i c l e
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Article history: Received 31 January 2017 Received in revised form 16 February 2017 Accepted 3 March 2017 Available online xxxx Keywords: Cyclosporine A Drug-induced gingival overgrowth Ligature-induced gingival inflammation
a b s t r a c t Drug-induced gingival overgrowth (DIGO) is a side effect of the enlargement of gingival tissue by phenytoin, nifedipine, and cyclosporine A (CsA). Gingival inflammation has been identified as a key factor that initiates DIGO. However, a sufficient animal model for clarifying the role of inflammation in DIGO has not yet been generated. We herein describe a novel CsA-induced gingival overgrowth mouse model to evaluate the role of inflammation. A ligature was placed around the second molar in maxillae for 7 days to induce gingival inflammation, and CsA (50 mg/kg/day) was administered to mice during each experimental period. The severity of gingival overgrowth and mRNA expression of inflammatory cytokines in gingiva were assessed by the gingival overgrowth degree, histological analyses, and RT-PCR. The administration of CsA for 28 days in combination with ligation significantly increased the gingival overgrowth degree and expanded the connective tissue area. Increases in the gingival overgrowth degree continued in a time-dependent manner until 21 days. Furthermore, the cessation of CsA reduced gingival overgrowth. Thin ligatures (7–0 size) induced weaker tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 mRNA expression and less gingival overgrowth than thick ligatures (5–0 ligature). Moreover, the administration of an antibiotic cocktail, which suppressed the expression of these inflammatory cytokines in gingiva, attenuated gingival overgrowth induced by ligatures and CsA. These results suggest that inflammation in gingival tissue plays a role in initiating CsA-induced gingival overgrowth. This gingival overgrowth mouse model has potential for elucidating the etiology of DIGO from the view point of gingival inflammation. © 2017 Published by Elsevier B.V.
1. Introduction Drug-induced gingival overgrowth (DIGO) generally occurs as a side effect of anti-convulsant drugs, i.e., phenytoin, calcium channel blockers, i.e., nifedipine, and immunosuppressants, i.e., cyclosporine A (CsA) (Trackman and Kantarci, 2004, 2015). DIGO patients are susceptible to gingival infections, inflammation, and disfiguration. Examples of the management of DIGO include oral hygiene, periodontal treatments, gingivectomy, and removal of the causative drugs. However, the recurrence of DIGO after gingivectomy and limitations in therapeutic methods are common in cases in which the removal of or alternations in drugs are impossible (Pernu et al., 1993; Khocht and Schneider, 1997; Bader et al., 1998). Therefore, novel therapy that provides an alternative to gingivectomy and drug alternations is needed.
⁎ Corresponding author at: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, 734-8553, Japan. E-mail address: [email protected] (S. Matsuda).
In the past decade, elevated levels of inflammatory cytokines in gingival tissue, including tumor-necrotizing factor-α (TNF-α), interleukin1β (IL-1β), and IL-6, have been regarded as a general explanation for the initiation and progression of DIGO because these cytokines facilitate fibroblast proliferation and extracellular matrix (ECM) protein production (Dill et al., 1993; Iacopino et al., 1997; James et al., 1998; Uzel et al., 2001). A previous clinical study demonstrated that reductions in gingival inflammation through oral hygiene ameliorated DIGO without alternations in the causative drugs (Pilatti and Sampaio, 1997). These findings imply the relevance of gingival inflammation in the context of DIGO. Therefore, elucidating the molecular mechanisms by which inflammation causes DIGO in more detail may lead to the establishment of a novel promising therapy. A dozen DIGO animal models, including dog, rabbit, and rat models, have been generated through extensive scientific efforts (Ishida et al., 1995; Nam et al., 2008; Jean et al., 2009); however, to the best of our knowledge, few DIGO mouse models currently exist. Mouse models are particularly informative when studying molecular events because of the number of genetically engineered strains available. Furthermore, a considerable amount of background information exists for gingival
http://dx.doi.org/10.1016/j.jim.2017.03.003 0022-1759/© 2017 Published by Elsevier B.V.
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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tissue and a wide range of immunological and cellular reagents are available. Abe and Hajishengallis (2013) recently optimized a ligature-induced periodontitis mouse model for the evaluation of periodontitis in a reliable and valid manner. Using this mouse model, we revealed that ligation on the second maxillary molar tooth elevated pro-inflammatory cytokine levels in gingival crevicular fluid (GCF), suggesting gingival inflammation (Matsuda et al., 2016). More specifically, this ligature-induced periodontitis model may be used to reliably clarify the role of inflammation in DIGO in mice. Thus, the aims of this study were 1) to establish a DIGO mouse model using ligature-induced periodontitis and the systemic administration of CsA and 2) to elucidate the possible etiological effects of inflammation on the onset of DIGO.
of the second molar and this ratio was multiplied by 100. This score was referred to as the gingival overgrowth degree (Fig. 1B). 2.4. Histological analysis Mouse maxillae were decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for 1 week and tissues were dehydrated through graded ethanol, cleared with xylene, and embedded in paraffin. Fivemicrometer-thick serial sections were cut in a buccal-palatal direction, stained with Hematoxylin and Eosin (HE), and observed under a light microscope for histomorphological evaluations. A quantitative analysis of the connective tissue area was employed according to a previous study (Spolidorio et al., 2005). 2.5. RT-PCR
2. Materials and methods 2.1. Ethics statement The smallest number of mice possible was used in each experiment. Mice were maintained in a vivarium, with the room temperature set at 22 ± 2 °C and a 12-h light/dark cycle (lights on/off at 8:00 AM/8:00 PM), and were given ad libitum access to food and water during the experimental period. All experiments utilizing animals were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals established by the Japanese Pharmacological Society and Hiroshima University, and procedures were reviewed and approved by the Committee of Research Facilities for Laboratory Animal Science of Hiroshima University. 2.2. Animals The experimental protocol described below was approved by the Animal Care Committee of Hiroshima University. Periodontitis was induced by a previously reported method (Abe and Hajishengallis, 2013). In order to induce gingival overgrowth, a sterile ligature (5–0 or 7–0 silk thread, Johnson & Johnson, NJ, USA) was placed around the maxillary second molar on both sides in C57BL/6j mice (8- to 12week-old males or females, Charles River Japan, Kanagawa, Japan) for 7 days before CsA (Tokyo Chemical Industry, Tokyo, Japan) or vehicle was intraperitoneally injected daily until day 28 (group #2, Fig. 1A). A time course study was conducted under the schedule of group #3 in Fig. 1A. Seven days after ligation, an evaluation was conducted every 7 days until day 28. In an attempt to clarify the effects of the cessation of CsA, CsA was withdrawn for 14 days after its administration (group #4, Fig. 1A). In order to investigate the role of inflammation in the onset of CsA, the placement of 7–0 ligatures (group #5, Fig. 1A) and treatment with antibiotics (group #6) were conducted. An antibiotic cocktail was administered at the beginning of ligation with 5–0 silk sutures for 14 days (group #6, Fig. 1A). CsA was diluted using 10% ethanol and 10% polyethyleneglycol castor oil (Wako Pure Chemical Industries) in sterilized distilled water up to a volume of 100%. CsA at a dosage of 50 mg/kg/day was used in all experiments. Mice with ligatures received drinking water containing an antibiotic cocktail (Rakoff-Nahoum et al., 2004) consisting of ampicillin (1 g/L, Sigma-Aldrich, St. Louis, MO), metronidazole (1 g/L, Sigma-Aldrich), vancomycin (0.5 g/L, Sigma-Aldrich), and neomycin sulfate (1 g/L, Sigma-Aldrich). 2.3. Macroscopic analysis Mice were sacrificed and the maxillae with gingiva and soft tissue were carefully collected. Images of gingiva on the occlusal surface were obtained using a stereomicroscope. In order to assess the severity of gingival overgrowth, we employed a previously reported method (Jean et al., 2009) with slight modifications. Briefly, we assessed the ratio of the gingival width (μm) on the buccal side to the width (μm)
RT-PCR was performed to examine the effects of silk suture sizes and antibiotics on inflammatory cytokine expression in gingiva. As shown in Fig. 1C, 7–0 and 5–0 silk sutures were ligated for 7 days (a), and antibiotics were administered for 14 days with ligatures (b). After sacrifice, palatal gingival tissue was extracted as previously described (Abe and Hajishengallis, 2013), then immediately placed in RNA later at 4 °C for 24 h before proceeding with the evaluation (Fig. 1C). Total RNA in gingival tissue (red-colored area) was isolated using an RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA). Extraction was performed according to the manufacturer's directions. A total of 500 ng of RNA was converted to cDNA using Sensiscript reverse transcriptase (QIAGEN). RT-PCR for the cloning of IL-1β was conducted for 28 cycles, while that for TNF-α and IL-6 was performed for 30 cycles of denaturation at 98 °C for 30 s and annealing and extension at 63 °C (IL-1β), 62 °C (IL-6), or 65 °C (TNF-α) for 30 s using Quick Taq® HS DyeMix (Toyobo., Osaka, Japan) and the following primers, which were made by referring to the sequences of mouse TNF-α: Forward; 5′CTCCCTCCAGAAAAGAGACCATGA-3′, Reverse; 5′-CTGACCACTCTCCCT TTGCAGAAC-3′, PCR products, 982 bp, IL-1β, Forward; 5′-GGATGATGA TGATAACCTGC-3′, Reverse 5′-CATGGAGAATATCACTTGTTGG-3′, PCR products, 163 bp and IL-6, forward; 5′-GAAATGAGAAAAGAGTTGT GCAATGG-3′, reverse; 5′–ATATCCAGTTTGGTAGCATCCATCAT-3′, PCR products, 123 bp. (Sigma-Aldrich) PCR images were recorded using a Bio-Rad ChemiDoc XRS image analysis system (Bio-Rad Laboratories, CA, USA). 2.6. Statistical analysis The Student's t-test was used for comparisons of two different outcomes from the experiments performed. A one-way ANOVA was performed followed by Tukey's test in order to assess differences in the severity of gingival overgrowth and the areas of connective tissue between ligation, non-ligation, CsA administration, and CsA with ligatures groups. 3. Results 3.1. Combination of ligatures with CsA induced gingival overgrowth in mice Four weeks after the administration of vehicle or CsA, gingival overgrowth was not observed in non-ligation group (group #1, Fig. 2A). Ligatures alone appeared to induce gingival swelling. However, ligatures with CsA apparently induced gingival overgrowth, which expanded around the ligatures and covered the second molar of the buccal side (group #2). In addition, ligatures with CsA significantly increased the gingival overgrowth degree more than the other conditions tested (Fig. 2B). Consistent with these macroscopic findings, histological evaluations demonstrated that the combination of ligatures and CsA induced a larger amount of dense connective tissue in the buccal gingiva than ligatures and vehicle (group #2, Fig. 2C). Furthermore, in higher
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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Fig. 1. Schematic presentation of the protocol used to develop the DIGO mouse model. (A) Schedule of experiments. Group #1; non-ligation group. CsA or vehicle was administered for 28 days without ligatures. Group #2; ligation group. Seven days after ligation, CsA or vehicle was intraperitoneally administered for 28 days. Group #3; time-course study. Mice were treated with CsA for 7, 14, 21, or 28 days with ligatures. Group #4; effect of CsA cessation. CsA was withdrawn for 14 days after its administration. Group #5; 7–0 ligation group. 7–0 silk sutures were ligated. The schedule was the same as that for group #2. Group #6; effect of an antibiotic treatment. Antibiotics were orally administered for 14 days at the beginning of ligation. (B) The gingival overgrowth degree was evaluated as the ratio of the gingival width (μm) on the buccal side to the width (μm) of the second molar and this ratio was multiplied by 100 (Gingival Overgrowth Degree). Bar = 1 mm. (C) Schedule for the analysis of the mRNA expression of inflammatory cytokines. 7–0 or 5–0 silk sutures were placed for 7 days (a) or antibiotics were administered for 14 days with ligation (b). The red-colored area was carefully extracted using a shaped scalpel for the isolation of RNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
magnification images, the expansion and density of fibers were greater in the CsA with ligatures group than in the other groups. The quantitative analysis of the connective tissue area also indicated that this area was significantly larger in the ligatures with CsA group than in the ligatures with vehicle group (group #2, Fig. 2D). The absence of ligation (group #1) did not affect periodontal tissue conditions. These results clearly indicate that the combination of ligatures and CsA induced gingival overgrowth in mice. A time-course study was conducted to clarify the role of the accumulation of CsA in the progression and maintenance of gingival overgrowth (group #3, Fig. 2E). Ligatures with the application of CsA increased the severity of gingival overgrowth in a time-dependent manner until 21 days and the degree of overgrowth was maintained at 28 days. However, ligatures with vehicle did not affect the gingival overgrowth degree until the end of the experimental period. We then tested the
effects of the cessation of CsA on the maintenance of gingival overgrowth (group #4, Fig. 2F). Fourteen days after the withdrawal of CsA, gingival overgrowth was significantly reduced to a similar level as that in the ligature alone group. These results suggest that the continuous application of CsA is indispensable for the progression and maintenance of gingival overgrowth. 3.2. Effects of inflammation on the initiation of gingival overgrowth In order to investigate the role of inflammation in the context of gingival overgrowth induced by the ligatures and CsA treatment, we compared the gingival overgrowth degree and inflammatory cytokine mRNA expression levels between CsA with thin (7–0 size) and thick (5–0 size) ligatures (groups #2 and #5). The gingival overgrowth degree was significantly lower in the CsA with 7–0 ligatures group than
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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Fig. 2. Combination of ligatures with CsA induced severe gingival overgrowth in mice. (A) Clinical photos of gingival tissue (Groups #1 and #2). (B) Gingival overgrowth degree at the endpoint of the experiment in groups #1 and #2 (mean ± SD, n = 8/group). **P b 0.01, significantly different from ligatures, a one-way ANOVA followed by Tukey's test. (C) HE staining of groups #1 and #2. At a lower magnification (×40), asterisks show ligatures in the gingival sulcus (group #2) and the square area was shown at a higher magnification (× 200). Bar = 200 μm at a lower magnification, Bar = 20 μm at a higher magnification. (D) The quantification of maxillary histomorphometric data (groups #1 and #2) was performed on 5 sections in each group. **P b 0.01, significantly different from ligatures, a one-way ANOVA followed by Tukey's test. (E) Results of the time-course study (group #3). CsA or vehicle was administered for the indicated time (mean ± SD, n = 8/group). **P b 0.01, significantly different from day 0, a one-way ANOVA followed by Tukey's test. †† P b 0.01, † P b 0.05, significantly different from vehicle at the same time point by the Student's t-test. (F) The effect of the withdrawal of CsA for 14 days (group #4). Mean ± SD, n = 8/group. **P b 0.01, significant difference between ligatures with CsA and ligatures alone or the withdrawal of CsA, a one-way ANOVA followed by Tukey's test.
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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in the CsA with 5–0 ligatures group (Fig. 3A). In accordance with this result, 7–0 ligatures induced IL-1β, TNF-α, and IL-6 mRNA expression; however, the mRNA expression levels of these inflammatory cytokine were markedly lower than those with 5–0 ligatures (Fig. 3B). We also investigated the effects of the antibiotic cocktail, which is considered to reduce oral bacterial infections in mice (Mawardi et al., 2011), on gingival overgrowth (groups #2 and #6). As expected, the antibiotic treatment ameliorated gingival overgrowth caused by the combination of ligatures and CsA (Fig. 3C). Consistent with these results, antibiotics also reduced inflammatory cytokine expression levels (Fig. 3D). These results indicate that ligature-induced inflammation plays a role in the onset of gingival overgrowth by CsA.
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4. Discussion In the present study, we developed a DIGO mouse model using ligation and CsA. This study was conducted following the experimental schedule shown in Fig. 1A. We demonstrated that the combination of ligation with CsA up-regulated the amount of connective tissue in gingiva (Fig. 2), and gingival overgrowth in this mouse model was alleviated by the cessation of the causative drug (Fig. 2D) or amelioration of inflammation (Fig. 3). These results are consistent with the symptoms of clinical DIGO, namely, alternations from CsA to other immunosuppressants (James et al., 2000) or the amelioration of inflammation by oral hygiene (Pilatti and Sampaio, 1997) attenuate gingival overgrowth. Accordingly,
Fig. 3. The role of ligature size and antibiotics on the development of DIGO. (A) Clinical photos and the gingival overgrowth degree with 7–0 and 5–0 ligatures and CsA (groups #2 and #5). Mean ± SD, n = 8/group. *P b 0.05 for the severity of gingival overgrowth. The Student's t-test. (B) mRNA expression of IL-1β, TNF-α, and IL-6. The schedule was shown in Fig. 1C (a). (C) The effect of antibiotics on the onset of gingival overgrowth (group #2 and #6). Mean ± SD, n = 8/group. *P b 0.05 for the severity of gingival overgrowth. The Student's t-test. (D) Cytokine expression in gingiva. The schedule was shown in Fig. 1C (b).
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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our DIGO mouse model reflects human clinical aspects, and, thus, may be a useful study tool for developing novel promising therapies for DIGO. Previous studies reported that drugs such as phenytoin or CsA induced gingival overgrowth in animals without periodontitis (Asahara et al., 2000; Meller et al., 2002; Assaggaf et al., 2015); however, in the present study, CsA alone failed to induce gingival overgrowth (Fig. 2). The long-term administration or a high and more frequent dosage schedule of CsA may have induced gingival overgrowth in our mice. In any case, it is important to note that gingival inflammation is a key factor in CsA-induced gingival overgrowth rather than phenytoin or nifedipine (Pilatti and Sampaio, 1997; Uzel et al., 2001; Guo et al., 2008; Suzuki et al., 2009; Fernandes et al., 2010; Trackman and Kantarci, 2015). Therefore, since a gingival overgrowth model induced by a causative drug alone lacks gingival inflammation, our DIGO model induced by ligation and CsA, which exhibited valid and severe gingival overgrowth, may be a better candidate for studies investigating the relationship between gingival inflammation and CsA. It is widely accepted that gingival inflammation caused by the placement of ligatures around molar teeth is associated with dysbiosis of the microbiome in the periodontal pocket, which results in the induction of host immune responses (Graves et al., 2008; Abe and Hajishengallis, 2013; Jiao et al., 2013). The results of the present study also showed that an antibiotic cocktail, which eliminates commensal bacteria (Rakoff-Nahoum et al., 2004; Mawardi et al., 2011), reduced gingival inflammation (Fig. 3B), suggesting that ligature-induced dysbiosis plays a role in the induction of inflammation in our DIGO mouse model. Therefore, a study targeting the microbiome or host immune response system may establish a novel promising therapy for DIGO. There is emerging evidence that inflammatory cytokine levels such as those of TNF-α, IL-1β, and IL-6 are up-regulated in the gingival tissue of DIGO patients (Williamson et al., 1994; Iacopino et al., 1997; Atilla and Kutukculer, 1998), and activation of the IL-1β and TNF-α targeting signaling molecule, nuclear factor kappa-B (NF-κB) has also been detected in the gingiva (Arabaci et al., 2014). However, the mechanisms by which this signaling molecule or cytokines play a critical role in the etiology of DIGO currently remain unclear. When used in conjunction with genetically engineered mice or a wide range of immunological and cellular reagents, the gingival overgrowth mouse model established in the present study may provide insights into the molecular mechanisms responsible for the etiology of DIGO. In conclusion, we herein demonstrated that the combination of ligature-induced gingival inflammation and CsA resulted in gingival overgrowth in mice. This model may facilitate further studies investigating the etiology of DIGO and the development of novel promising therapies. Acknowledgments This study was supported in part by a Grant-in-Aid for Scientific Research (B) (25670885) and a Grant-in-Aid for the Encouragement of Young Scientists (B) (26861815) from the Japan Society for the Promotion of Science, Japan. This manuscript was proofread by Medical English Service. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this manuscript. References Abe, T., Hajishengallis, G., 2013. Optimization of the ligature-induced periodontitis model in mice. J. Immunol. Methods 394, 49–54. Arabaci, T., Kose, O., Kizildag, A., Albayrak, M., Cicek, Y., Kara, A., 2014. Role of nuclear factor kappa-B in phenytoin-induced gingival overgrowth. Oral Dis. 20, 294–300. Asahara, Y., Nishimura, F., Yamada, H., Naruishi, K., Kataoka, M., Kido, J., Nagata, T., Murayama, Y., 2000. Mast cells are not involved in the development of cyclosporin
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Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation Ai Okanobu a, Shinji Matsuda a,⁎, Mikihito Kajiya a, Tsuyoshi Fujita a, Mizuho Kittaka a, Hideki Shiba b, Hidemi Kurihara a a b
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Japan Department of Biological Endodontics, Integrated Health Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Japan
a r t i c l e
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Article history: Received 31 January 2017 Received in revised form 16 February 2017 Accepted 3 March 2017 Available online xxxx Keywords: Cyclosporine A Drug-induced gingival overgrowth Ligature-induced gingival inflammation
a b s t r a c t Drug-induced gingival overgrowth (DIGO) is a side effect of the enlargement of gingival tissue by phenytoin, nifedipine, and cyclosporine A (CsA). Gingival inflammation has been identified as a key factor that initiates DIGO. However, a sufficient animal model for clarifying the role of inflammation in DIGO has not yet been generated. We herein describe a novel CsA-induced gingival overgrowth mouse model to evaluate the role of inflammation. A ligature was placed around the second molar in maxillae for 7 days to induce gingival inflammation, and CsA (50 mg/kg/day) was administered to mice during each experimental period. The severity of gingival overgrowth and mRNA expression of inflammatory cytokines in gingiva were assessed by the gingival overgrowth degree, histological analyses, and RT-PCR. The administration of CsA for 28 days in combination with ligation significantly increased the gingival overgrowth degree and expanded the connective tissue area. Increases in the gingival overgrowth degree continued in a time-dependent manner until 21 days. Furthermore, the cessation of CsA reduced gingival overgrowth. Thin ligatures (7–0 size) induced weaker tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 mRNA expression and less gingival overgrowth than thick ligatures (5–0 ligature). Moreover, the administration of an antibiotic cocktail, which suppressed the expression of these inflammatory cytokines in gingiva, attenuated gingival overgrowth induced by ligatures and CsA. These results suggest that inflammation in gingival tissue plays a role in initiating CsA-induced gingival overgrowth. This gingival overgrowth mouse model has potential for elucidating the etiology of DIGO from the view point of gingival inflammation. © 2017 Published by Elsevier B.V.
1. Introduction Drug-induced gingival overgrowth (DIGO) generally occurs as a side effect of anti-convulsant drugs, i.e., phenytoin, calcium channel blockers, i.e., nifedipine, and immunosuppressants, i.e., cyclosporine A (CsA) (Trackman and Kantarci, 2004, 2015). DIGO patients are susceptible to gingival infections, inflammation, and disfiguration. Examples of the management of DIGO include oral hygiene, periodontal treatments, gingivectomy, and removal of the causative drugs. However, the recurrence of DIGO after gingivectomy and limitations in therapeutic methods are common in cases in which the removal of or alternations in drugs are impossible (Pernu et al., 1993; Khocht and Schneider, 1997; Bader et al., 1998). Therefore, novel therapy that provides an alternative to gingivectomy and drug alternations is needed.
⁎ Corresponding author at: Department of Periodontal Medicine, Division of Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, 734-8553, Japan. E-mail address: [email protected] (S. Matsuda).
In the past decade, elevated levels of inflammatory cytokines in gingival tissue, including tumor-necrotizing factor-α (TNF-α), interleukin1β (IL-1β), and IL-6, have been regarded as a general explanation for the initiation and progression of DIGO because these cytokines facilitate fibroblast proliferation and extracellular matrix (ECM) protein production (Dill et al., 1993; Iacopino et al., 1997; James et al., 1998; Uzel et al., 2001). A previous clinical study demonstrated that reductions in gingival inflammation through oral hygiene ameliorated DIGO without alternations in the causative drugs (Pilatti and Sampaio, 1997). These findings imply the relevance of gingival inflammation in the context of DIGO. Therefore, elucidating the molecular mechanisms by which inflammation causes DIGO in more detail may lead to the establishment of a novel promising therapy. A dozen DIGO animal models, including dog, rabbit, and rat models, have been generated through extensive scientific efforts (Ishida et al., 1995; Nam et al., 2008; Jean et al., 2009); however, to the best of our knowledge, few DIGO mouse models currently exist. Mouse models are particularly informative when studying molecular events because of the number of genetically engineered strains available. Furthermore, a considerable amount of background information exists for gingival
http://dx.doi.org/10.1016/j.jim.2017.03.003 0022-1759/© 2017 Published by Elsevier B.V.
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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tissue and a wide range of immunological and cellular reagents are available. Abe and Hajishengallis (2013) recently optimized a ligature-induced periodontitis mouse model for the evaluation of periodontitis in a reliable and valid manner. Using this mouse model, we revealed that ligation on the second maxillary molar tooth elevated pro-inflammatory cytokine levels in gingival crevicular fluid (GCF), suggesting gingival inflammation (Matsuda et al., 2016). More specifically, this ligature-induced periodontitis model may be used to reliably clarify the role of inflammation in DIGO in mice. Thus, the aims of this study were 1) to establish a DIGO mouse model using ligature-induced periodontitis and the systemic administration of CsA and 2) to elucidate the possible etiological effects of inflammation on the onset of DIGO.
of the second molar and this ratio was multiplied by 100. This score was referred to as the gingival overgrowth degree (Fig. 1B). 2.4. Histological analysis Mouse maxillae were decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for 1 week and tissues were dehydrated through graded ethanol, cleared with xylene, and embedded in paraffin. Fivemicrometer-thick serial sections were cut in a buccal-palatal direction, stained with Hematoxylin and Eosin (HE), and observed under a light microscope for histomorphological evaluations. A quantitative analysis of the connective tissue area was employed according to a previous study (Spolidorio et al., 2005). 2.5. RT-PCR
2. Materials and methods 2.1. Ethics statement The smallest number of mice possible was used in each experiment. Mice were maintained in a vivarium, with the room temperature set at 22 ± 2 °C and a 12-h light/dark cycle (lights on/off at 8:00 AM/8:00 PM), and were given ad libitum access to food and water during the experimental period. All experiments utilizing animals were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals established by the Japanese Pharmacological Society and Hiroshima University, and procedures were reviewed and approved by the Committee of Research Facilities for Laboratory Animal Science of Hiroshima University. 2.2. Animals The experimental protocol described below was approved by the Animal Care Committee of Hiroshima University. Periodontitis was induced by a previously reported method (Abe and Hajishengallis, 2013). In order to induce gingival overgrowth, a sterile ligature (5–0 or 7–0 silk thread, Johnson & Johnson, NJ, USA) was placed around the maxillary second molar on both sides in C57BL/6j mice (8- to 12week-old males or females, Charles River Japan, Kanagawa, Japan) for 7 days before CsA (Tokyo Chemical Industry, Tokyo, Japan) or vehicle was intraperitoneally injected daily until day 28 (group #2, Fig. 1A). A time course study was conducted under the schedule of group #3 in Fig. 1A. Seven days after ligation, an evaluation was conducted every 7 days until day 28. In an attempt to clarify the effects of the cessation of CsA, CsA was withdrawn for 14 days after its administration (group #4, Fig. 1A). In order to investigate the role of inflammation in the onset of CsA, the placement of 7–0 ligatures (group #5, Fig. 1A) and treatment with antibiotics (group #6) were conducted. An antibiotic cocktail was administered at the beginning of ligation with 5–0 silk sutures for 14 days (group #6, Fig. 1A). CsA was diluted using 10% ethanol and 10% polyethyleneglycol castor oil (Wako Pure Chemical Industries) in sterilized distilled water up to a volume of 100%. CsA at a dosage of 50 mg/kg/day was used in all experiments. Mice with ligatures received drinking water containing an antibiotic cocktail (Rakoff-Nahoum et al., 2004) consisting of ampicillin (1 g/L, Sigma-Aldrich, St. Louis, MO), metronidazole (1 g/L, Sigma-Aldrich), vancomycin (0.5 g/L, Sigma-Aldrich), and neomycin sulfate (1 g/L, Sigma-Aldrich). 2.3. Macroscopic analysis Mice were sacrificed and the maxillae with gingiva and soft tissue were carefully collected. Images of gingiva on the occlusal surface were obtained using a stereomicroscope. In order to assess the severity of gingival overgrowth, we employed a previously reported method (Jean et al., 2009) with slight modifications. Briefly, we assessed the ratio of the gingival width (μm) on the buccal side to the width (μm)
RT-PCR was performed to examine the effects of silk suture sizes and antibiotics on inflammatory cytokine expression in gingiva. As shown in Fig. 1C, 7–0 and 5–0 silk sutures were ligated for 7 days (a), and antibiotics were administered for 14 days with ligatures (b). After sacrifice, palatal gingival tissue was extracted as previously described (Abe and Hajishengallis, 2013), then immediately placed in RNA later at 4 °C for 24 h before proceeding with the evaluation (Fig. 1C). Total RNA in gingival tissue (red-colored area) was isolated using an RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA). Extraction was performed according to the manufacturer's directions. A total of 500 ng of RNA was converted to cDNA using Sensiscript reverse transcriptase (QIAGEN). RT-PCR for the cloning of IL-1β was conducted for 28 cycles, while that for TNF-α and IL-6 was performed for 30 cycles of denaturation at 98 °C for 30 s and annealing and extension at 63 °C (IL-1β), 62 °C (IL-6), or 65 °C (TNF-α) for 30 s using Quick Taq® HS DyeMix (Toyobo., Osaka, Japan) and the following primers, which were made by referring to the sequences of mouse TNF-α: Forward; 5′CTCCCTCCAGAAAAGAGACCATGA-3′, Reverse; 5′-CTGACCACTCTCCCT TTGCAGAAC-3′, PCR products, 982 bp, IL-1β, Forward; 5′-GGATGATGA TGATAACCTGC-3′, Reverse 5′-CATGGAGAATATCACTTGTTGG-3′, PCR products, 163 bp and IL-6, forward; 5′-GAAATGAGAAAAGAGTTGT GCAATGG-3′, reverse; 5′–ATATCCAGTTTGGTAGCATCCATCAT-3′, PCR products, 123 bp. (Sigma-Aldrich) PCR images were recorded using a Bio-Rad ChemiDoc XRS image analysis system (Bio-Rad Laboratories, CA, USA). 2.6. Statistical analysis The Student's t-test was used for comparisons of two different outcomes from the experiments performed. A one-way ANOVA was performed followed by Tukey's test in order to assess differences in the severity of gingival overgrowth and the areas of connective tissue between ligation, non-ligation, CsA administration, and CsA with ligatures groups. 3. Results 3.1. Combination of ligatures with CsA induced gingival overgrowth in mice Four weeks after the administration of vehicle or CsA, gingival overgrowth was not observed in non-ligation group (group #1, Fig. 2A). Ligatures alone appeared to induce gingival swelling. However, ligatures with CsA apparently induced gingival overgrowth, which expanded around the ligatures and covered the second molar of the buccal side (group #2). In addition, ligatures with CsA significantly increased the gingival overgrowth degree more than the other conditions tested (Fig. 2B). Consistent with these macroscopic findings, histological evaluations demonstrated that the combination of ligatures and CsA induced a larger amount of dense connective tissue in the buccal gingiva than ligatures and vehicle (group #2, Fig. 2C). Furthermore, in higher
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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Fig. 1. Schematic presentation of the protocol used to develop the DIGO mouse model. (A) Schedule of experiments. Group #1; non-ligation group. CsA or vehicle was administered for 28 days without ligatures. Group #2; ligation group. Seven days after ligation, CsA or vehicle was intraperitoneally administered for 28 days. Group #3; time-course study. Mice were treated with CsA for 7, 14, 21, or 28 days with ligatures. Group #4; effect of CsA cessation. CsA was withdrawn for 14 days after its administration. Group #5; 7–0 ligation group. 7–0 silk sutures were ligated. The schedule was the same as that for group #2. Group #6; effect of an antibiotic treatment. Antibiotics were orally administered for 14 days at the beginning of ligation. (B) The gingival overgrowth degree was evaluated as the ratio of the gingival width (μm) on the buccal side to the width (μm) of the second molar and this ratio was multiplied by 100 (Gingival Overgrowth Degree). Bar = 1 mm. (C) Schedule for the analysis of the mRNA expression of inflammatory cytokines. 7–0 or 5–0 silk sutures were placed for 7 days (a) or antibiotics were administered for 14 days with ligation (b). The red-colored area was carefully extracted using a shaped scalpel for the isolation of RNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
magnification images, the expansion and density of fibers were greater in the CsA with ligatures group than in the other groups. The quantitative analysis of the connective tissue area also indicated that this area was significantly larger in the ligatures with CsA group than in the ligatures with vehicle group (group #2, Fig. 2D). The absence of ligation (group #1) did not affect periodontal tissue conditions. These results clearly indicate that the combination of ligatures and CsA induced gingival overgrowth in mice. A time-course study was conducted to clarify the role of the accumulation of CsA in the progression and maintenance of gingival overgrowth (group #3, Fig. 2E). Ligatures with the application of CsA increased the severity of gingival overgrowth in a time-dependent manner until 21 days and the degree of overgrowth was maintained at 28 days. However, ligatures with vehicle did not affect the gingival overgrowth degree until the end of the experimental period. We then tested the
effects of the cessation of CsA on the maintenance of gingival overgrowth (group #4, Fig. 2F). Fourteen days after the withdrawal of CsA, gingival overgrowth was significantly reduced to a similar level as that in the ligature alone group. These results suggest that the continuous application of CsA is indispensable for the progression and maintenance of gingival overgrowth. 3.2. Effects of inflammation on the initiation of gingival overgrowth In order to investigate the role of inflammation in the context of gingival overgrowth induced by the ligatures and CsA treatment, we compared the gingival overgrowth degree and inflammatory cytokine mRNA expression levels between CsA with thin (7–0 size) and thick (5–0 size) ligatures (groups #2 and #5). The gingival overgrowth degree was significantly lower in the CsA with 7–0 ligatures group than
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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Fig. 2. Combination of ligatures with CsA induced severe gingival overgrowth in mice. (A) Clinical photos of gingival tissue (Groups #1 and #2). (B) Gingival overgrowth degree at the endpoint of the experiment in groups #1 and #2 (mean ± SD, n = 8/group). **P b 0.01, significantly different from ligatures, a one-way ANOVA followed by Tukey's test. (C) HE staining of groups #1 and #2. At a lower magnification (×40), asterisks show ligatures in the gingival sulcus (group #2) and the square area was shown at a higher magnification (× 200). Bar = 200 μm at a lower magnification, Bar = 20 μm at a higher magnification. (D) The quantification of maxillary histomorphometric data (groups #1 and #2) was performed on 5 sections in each group. **P b 0.01, significantly different from ligatures, a one-way ANOVA followed by Tukey's test. (E) Results of the time-course study (group #3). CsA or vehicle was administered for the indicated time (mean ± SD, n = 8/group). **P b 0.01, significantly different from day 0, a one-way ANOVA followed by Tukey's test. †† P b 0.01, † P b 0.05, significantly different from vehicle at the same time point by the Student's t-test. (F) The effect of the withdrawal of CsA for 14 days (group #4). Mean ± SD, n = 8/group. **P b 0.01, significant difference between ligatures with CsA and ligatures alone or the withdrawal of CsA, a one-way ANOVA followed by Tukey's test.
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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in the CsA with 5–0 ligatures group (Fig. 3A). In accordance with this result, 7–0 ligatures induced IL-1β, TNF-α, and IL-6 mRNA expression; however, the mRNA expression levels of these inflammatory cytokine were markedly lower than those with 5–0 ligatures (Fig. 3B). We also investigated the effects of the antibiotic cocktail, which is considered to reduce oral bacterial infections in mice (Mawardi et al., 2011), on gingival overgrowth (groups #2 and #6). As expected, the antibiotic treatment ameliorated gingival overgrowth caused by the combination of ligatures and CsA (Fig. 3C). Consistent with these results, antibiotics also reduced inflammatory cytokine expression levels (Fig. 3D). These results indicate that ligature-induced inflammation plays a role in the onset of gingival overgrowth by CsA.
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4. Discussion In the present study, we developed a DIGO mouse model using ligation and CsA. This study was conducted following the experimental schedule shown in Fig. 1A. We demonstrated that the combination of ligation with CsA up-regulated the amount of connective tissue in gingiva (Fig. 2), and gingival overgrowth in this mouse model was alleviated by the cessation of the causative drug (Fig. 2D) or amelioration of inflammation (Fig. 3). These results are consistent with the symptoms of clinical DIGO, namely, alternations from CsA to other immunosuppressants (James et al., 2000) or the amelioration of inflammation by oral hygiene (Pilatti and Sampaio, 1997) attenuate gingival overgrowth. Accordingly,
Fig. 3. The role of ligature size and antibiotics on the development of DIGO. (A) Clinical photos and the gingival overgrowth degree with 7–0 and 5–0 ligatures and CsA (groups #2 and #5). Mean ± SD, n = 8/group. *P b 0.05 for the severity of gingival overgrowth. The Student's t-test. (B) mRNA expression of IL-1β, TNF-α, and IL-6. The schedule was shown in Fig. 1C (a). (C) The effect of antibiotics on the onset of gingival overgrowth (group #2 and #6). Mean ± SD, n = 8/group. *P b 0.05 for the severity of gingival overgrowth. The Student's t-test. (D) Cytokine expression in gingiva. The schedule was shown in Fig. 1C (b).
Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003
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our DIGO mouse model reflects human clinical aspects, and, thus, may be a useful study tool for developing novel promising therapies for DIGO. Previous studies reported that drugs such as phenytoin or CsA induced gingival overgrowth in animals without periodontitis (Asahara et al., 2000; Meller et al., 2002; Assaggaf et al., 2015); however, in the present study, CsA alone failed to induce gingival overgrowth (Fig. 2). The long-term administration or a high and more frequent dosage schedule of CsA may have induced gingival overgrowth in our mice. In any case, it is important to note that gingival inflammation is a key factor in CsA-induced gingival overgrowth rather than phenytoin or nifedipine (Pilatti and Sampaio, 1997; Uzel et al., 2001; Guo et al., 2008; Suzuki et al., 2009; Fernandes et al., 2010; Trackman and Kantarci, 2015). Therefore, since a gingival overgrowth model induced by a causative drug alone lacks gingival inflammation, our DIGO model induced by ligation and CsA, which exhibited valid and severe gingival overgrowth, may be a better candidate for studies investigating the relationship between gingival inflammation and CsA. It is widely accepted that gingival inflammation caused by the placement of ligatures around molar teeth is associated with dysbiosis of the microbiome in the periodontal pocket, which results in the induction of host immune responses (Graves et al., 2008; Abe and Hajishengallis, 2013; Jiao et al., 2013). The results of the present study also showed that an antibiotic cocktail, which eliminates commensal bacteria (Rakoff-Nahoum et al., 2004; Mawardi et al., 2011), reduced gingival inflammation (Fig. 3B), suggesting that ligature-induced dysbiosis plays a role in the induction of inflammation in our DIGO mouse model. Therefore, a study targeting the microbiome or host immune response system may establish a novel promising therapy for DIGO. There is emerging evidence that inflammatory cytokine levels such as those of TNF-α, IL-1β, and IL-6 are up-regulated in the gingival tissue of DIGO patients (Williamson et al., 1994; Iacopino et al., 1997; Atilla and Kutukculer, 1998), and activation of the IL-1β and TNF-α targeting signaling molecule, nuclear factor kappa-B (NF-κB) has also been detected in the gingiva (Arabaci et al., 2014). However, the mechanisms by which this signaling molecule or cytokines play a critical role in the etiology of DIGO currently remain unclear. When used in conjunction with genetically engineered mice or a wide range of immunological and cellular reagents, the gingival overgrowth mouse model established in the present study may provide insights into the molecular mechanisms responsible for the etiology of DIGO. In conclusion, we herein demonstrated that the combination of ligature-induced gingival inflammation and CsA resulted in gingival overgrowth in mice. This model may facilitate further studies investigating the etiology of DIGO and the development of novel promising therapies. Acknowledgments This study was supported in part by a Grant-in-Aid for Scientific Research (B) (25670885) and a Grant-in-Aid for the Encouragement of Young Scientists (B) (26861815) from the Japan Society for the Promotion of Science, Japan. This manuscript was proofread by Medical English Service. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this manuscript. References Abe, T., Hajishengallis, G., 2013. Optimization of the ligature-induced periodontitis model in mice. J. Immunol. Methods 394, 49–54. Arabaci, T., Kose, O., Kizildag, A., Albayrak, M., Cicek, Y., Kara, A., 2014. Role of nuclear factor kappa-B in phenytoin-induced gingival overgrowth. Oral Dis. 20, 294–300. Asahara, Y., Nishimura, F., Yamada, H., Naruishi, K., Kataoka, M., Kido, J., Nagata, T., Murayama, Y., 2000. Mast cells are not involved in the development of cyclosporin
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Please cite this article as: Okanobu, A., et al., A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation, J. Immunol. Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.03.003