Ligature-induced Periodontitis Model Research Articles

DC-STAMP Is an Osteoclast Fusogen Engaged in Periodontal Bone Resorption

Dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in the induction of osteoclast (OC) cell fusion, as well as DC-mediated immune regulation. While DC-STAMP gene expression is upregulated in the gingival tissue with periodontitis, its pathophysiological roles in periodontitis remain unclear. To evaluate the effects of DC-STAMP in periodontitis, anti-DC-STAMP–monoclonal antibody (mAb) was tested in a mouse model of ligature-induced periodontitis (n = 6–7/group) where Pasteurella pneumotropica (Pp)-reactive immune response activated T cells to produce receptor activator of nuclear factor kappa-B ligand (RANKL), which, in turn, promotes the periodontal bone loss via upregulation of osteoclastogenesis. DC-STAMP was expressed on the cell surface of mature multinuclear OCs, as well as immature mononuclear OCs, in primary cultures of RANKL-stimulated bone marrow cells. Anti-DC-STAMP-mAb suppressed the emergence of large, but not small, multinuclear OCs, suggesting that DC-STAMP is engaged in the late stage of cell fusion. Anti-DC-STAMP-mAb also inhibited pit formation caused by RANKL-stimulated bone marrow cells. Attachment of ligature to a second maxillary molar induced DC-STAMP messenger RNA and protein, along with elevated tartrate-resistant acid phosphatase–positive (TRAP+) OCs and alveolar bone loss. As we expected, systemic administration of anti-DC-STAMP-mAb downregulated the ligature-induced alveolar bone loss. Importantly, local injection of anti-DC-STAMP-mAb also suppressed alveolar bone loss and reduced the total number of multinucleated TRAP+ cells in mice that received ligature attachment. Attachment of ligature induced significantly elevated tumor necrosis factor–α, interleukin-1β, and RANKL in the gingival tissue compared with the control site without ligature (P < 0.05), which was unaffected by local injection with either anti-DC-STAMP-mAb or control-mAb. Neither in vivo anti-Pp IgG antibody nor in vitro anti-Pp T-cell response and resultant production of RANKL was affected by anti-DC-STAMP-mAb. This study illustrated the roles of DC-STAMP in promoting local OC cell fusion without affecting adaptive immune responses to oral bacteria. Therefore, it is plausible that a novel therapeutic regimen targeting DC-STAMP could suppress periodontal bone loss.

Effect of local bone marrow stromal cell administration on ligature-induced periodontitis in mice.

Bone marrow-derived multipotent stromal cells (BMSCs) have potent antiinflammatory effects. This study aimed to investigate the antiinflammatory potential of BMSCs using a mouse model of ligature-induced periodontitis. BMSCs were isolated from the femurs and tibiae of mice. Periodontitis was induced by placing a ligature around the right maxillary second molar. After 3 days, the mice were administered BMSC in the gingiva of the mesial interdental papilla around the ligatured molar. The ligatured and non-ligatured mice that were not administered BMSC served as controls. Differences in inflammatory infiltration and bone resorption around the roots of the second molar were assessed and were subsequently quantified using microcomputed tomography (micro-CT), histological analysis, and tartrate-resistant acid phosphatase (TRAP) staining. Micro-CT revealed that alveolar bone loss around the ligatured molars increased in a time-dependent manner; however, the effect was significantly less in BMSC-treated mice compared with ligatured control mice. Tissue histopathology revealed that BMSC administration mitigated inflammatory infiltration in ligatured BMSC mice. In addition, the number of TRAP-positive osteoclasts was markedly elevated in ligatured control mice compared with those in BMSC-treated mice. These findings indicate that local BMSC administration can mitigate inflammation and alveolar bone resorption, suggesting that administering BMSC leads to new therapeutics for periodontitis.

Bone metabolic microarray analysis of ligature-induced periodontitis in streptozotocin-induced diabetic mice.

Periodontal disease is a chronic infectious disease that results in bone loss. Many epidemiological studies have reported the progression of periodontal tissue destruction in patients with diabetes; however, the associated mechanism remains unclear. In this study, we comprehensively investigated how diabetes affects the periodontal tissue and alveolar bone loss using a ligature-induced periodontitis model in streptozotocin-induced diabetic (STZ) mice. Diabetes was induced by intraperitoneal injection with streptozotocin in 6-wk-old C57/BL6J male mice. A silk ligature was tied around the maxillary left second molar in 9-wk-old wild-type (WT) and STZ mice. Bone loss was evaluated at 3 and 7 d after ligation. mRNA expression levels in the gingiva between the two groups were examined by DNA microarray and quantitative polymerase chain reaction at 1, 3 and 7 d post-ligation. Tartrate-resistant acid phosphatase and alkaline phosphatase staining of the periodontal tissue was performed for evaluation of osteoclasts and osteoblasts in histological analysis. In the gingiva, hyperglycemia upregulated the osteoprotegerin (Opg) mRNA expression and downregulated Osteocalcin mRNA expression. In the ligated gingiva, tumor necrosis factor-α (Tnf-α) mRNA expression was upregulated at 1 d post-ligation in STZ mice but not in WT mice. At 3 d post-ligation, alveolar bone loss was observed in STZ mice, but not in WT mice. Significantly severe alveolar bone loss was observed in STZ mice compared to WT mice at 7 d post-ligation. Bone metabolic analysis using DNA microarray showed significant downregulation in the mRNA expression of glioma-associated oncogene homologue 1 (Gli1) and collagen type VI alpha 1 (Col6a1) at the gingiva of the ligated site in STZ mice compared to that in WT mice. Quantitative polymerase chain reaction showed that Gli1 and Col6a1 mRNA expression levels were significantly downregulated in the gingiva of the ligated site in STZ mice compared to WT mice. Histological analysis showed lower alkaline phosphatase activity in STZ mice. In addition, an increased number of tartrate-resistant acid phosphatase-positive multinucleated cells were observed at the ligated sites in STZ mice. These results suggest that an imbalance of bone metabolism causes osteoclastosis in insulin-deficient diabetes, and that alveolar bone loss could occur at an early phase under this condition.

Ligature-induced periodontitis in mice induces elevated levels of circulating interleukin-6 but shows only weak effects on adipose and liver tissues.

Our previous study demonstrated using an oral gavage model that Porphyromonas gingivalis could induce various inflammatory changes linked to periodontitis-associated systemic diseases by altering gut microbiota. A ligature-induced periodontitis model is similar to human periodontitis in various aspects: in both cases, alveolar bone resorption depends on oral bacterial load, and gingival tissue becomes infiltrated with inflammatory cells. Therefore, this model may be suitable for the analysis of bacterial burden and gingival tissue inflammation with changes related to systemic diseases. Periodontal tissue destruction was induced by a 2 wk ligature placement around the bilateral maxillary second molar. We analyzed the expression profile of various genes in several tissues, levels of systemic inflammatory markers and induction of insulin resistance. In addition, we studied changes in gut microbiota composition and bacterial load in the oral cavity. Two weeks after ligature placement gingival inflammation was significantly induced with a disrupted gingival epithelial barrier and alveolar bone resorption accompanied by increased bacterial burden in the oral cavity. Gene expression analysis of the gingival tissue of ligated mice demonstrated that interleukin (Il)1b was significantly elevated and Il6 and Il17a tended to be higher in ligated mice than in untreated mice. Although serum IL-6 was significantly elevated and serum amyloid A tended to be higher in ligated compared to untreated mice, endotoxin levels did not differ between the two groups. Among the genes whose expressions are closely related to glucose and lipid metabolisms, only phosphoenolpyruvate carboxykinase 1 (Pck1) and acetyl-coenzyme A carboxylase alpha (Acaca) showed significant changes following ligature placement in the liver, with the former upregulated and the latter downregulated. However, insulin sensitivity did not change following ligature placement. Furthermore, ligature placement weakly affected the composition of gut microbiota and gene expression in the intestines. The results suggest that increased oral commensals and gingival inflammation have limited roles in the pathological changes to adipose and liver tissues, which are important organs whose dysfunctions contribute to the development of periodontitis-related systemic diseases.

Nanotechnology in Phytotherapy: Antiinflammatory Effect of a Nanostructured Thymol Gel from Lippia sidoides in Acute Periodontitis in Rats.

Lippia sidoides Cham (Verbenaceae) is largely distributed in the northeastern region of Brazil. It is popularly known as 'Alecrim-pimenta'. Recent studies have shown that some species of Lippia have interesting pharmacological activities. This study aimed to evaluate the effect of a nanostructured thymol gel (TG) 1.2 mg/g on acute phase of ligature-induced periodontitis model [acute periodontal disease (APD)] in rats. APD was induced in 24 Wistar rats subjected to ligature placement on left molars in maxillae. Animals were treated with TG, immediately after APD induction. Saline-based gel was utilized as negative control and diethylammonium diclofenac gel 10 mg/g was used as positive control. Animals were randomly assigned into the groups. The periodontium and the surrounding gingiva were examined at histopathology, as well as the neutrophil influx into the gingiva was assayed using myeloperoxidase activity levels by ELISA method. TG treatment reduced tissue lesion at histopathology coupled to decreased myeloperoxidase activity production in gingival tissue when compared with the saline gel control group (p < 0.05). The TG gel was able to provide a significant myeloperoxidase decreasing in gingiva tissue confirming to be effective in reducing gingival inflammation in this model.

Eugenol nanocapsule for enhanced therapeutic activity against periodontal infections

Eugenol is a godsend to dental care due to its analgesic, local anesthetic, and anti-inflammatory and antibacterial effects. The aim of the present research work was to prepare, characterize and evaluate eugenol-loaded nanocapsules (NCs) against periodontal infections. Eugenol-loaded polycaprolactone (PCL) NCs were prepared by solvent displacement method. The nanometric size of the prepared NCs was confirmed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The in vitro drug release was found to follow a biphasic pattern and followed Michaelis–Menten like model. The percentage cell viability values near to 100 in the cell viability assay indicated that the NCs are not cytotoxic. In the in vivo studies, the eugenol NC group displayed significant difference in the continuity of epithelium of the interdental papilla in comparison to the untreated, pure eugenol and placebo groups. The in vivo performance of the eugenol-loaded NCs using ligature-induced periodontitis model in rats indicated that eugenol-loaded NCs could prevent septal bone resorption in periodontitis. On the basis of our research findings it could be concluded that eugenol-loaded PCL NCs could serve as a novel colloidal drug delivery system for enhanced therapeutic activity of eugenol in the treatment of periodontal infections.

Puerarin decreases bone loss and collagen destruction in rats with ligature-induced periodontitis.

Puerarin, the most abundant isoflavonoid in kudzu root, shows various bioactivities, including bone-sparing, anti-inflammatory and antiproteinase properties. This study aimed to evaluate the effects of puerarin in a rat model of ligature-induced periodontitis. Rat models of periodontitis were developed by bilaterally placing ligatures around the first mandibular molars. Puerarin was administrated daily by gavage at doses of 100, 200 and 400 mg/kg, starting a day before the placement of ligatures. Rats were humanely killed 7 d after the induction of periodontitis. Micro-computed tomography and sirius red staining were used to evaluate alveolar bone loss and collagen destruction, respectively. Histomorphometrical analysis was used to assess the inflammatory cell infiltration. Immunohistochemistry and tartrate-resistant acid phosphatase were used to detect receptor activator of nuclear factor kappa B ligand and osteoprotegerin expressions, and osteoclast activity in the gingiva and alveolar bone. The activation of nuclear factor-kappa B, production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, glycosylation of extracellular matrix metalloproteinase inducer, and production of matrix metalloproteinase (MMP)-2 and MMP-9 in the gingiva were assessed by Western blot. Puerarin at doses of 200 and 400 mg/kg significantly reduced the alveolar bone loss compared with the vehicle group. Collagen destruction and inflammatory cell infiltration were significantly less in the puerarin-treated group (200 mg/kg) compared with that of the vehicle group. Puerarin (200 mg/kg) also reduced the ratio of receptor activator of nuclear factor kappa B ligand/osteoprotegerin and osteoclast activity. Western blot analysis showed that puerarin (200 mg/kg) inhibited the activation of nuclear factor-kappa B p65, which is associated with lower IL-1β and TNF-α production, and reduced the glycosylation of extracellular matrix metalloproteinase inducer, which is associated with lower levels of MMP-2 and MMP-9. Puerarin reduced the alveolar bone loss and collagen destruction in rats with ligature-induced periodontitis by inhibiting the production of RANKL, IL-1β, TNF-α, MMP-2 and MMP-9. Thus, puerarin can be considered a promising agent for adjunctive therapy of periodontitis.

Topical application of herbal formula for the treatment of ligature-induced periodontitis.

The aim of this study was to investigate the therapeutic effects of a herbal formula, PerioH-035, containing Angelica sinensis, steamed Rehmannia glutinosa, Angelica dahurica, Cimicifuga heracleifolia, and Zanthoxylum piperitum on the periodontal breakdown in a well-established ligature-induced periodontitis model in rats. Sprague-Dawley rats were randomly assigned to 1 of 4 groups: NL (non-ligatured), L (ligatured), P1 (ligatured and treated with 1 mg/mL PerioH-035), P100 (ligatured and treated with 100 mg/mL PerioH-035). Periodontitis was induced by placing a ligature around the mandibular first molars. PerioH-035 was topically applied to both sides of the first molar for 2 weeks. The right side of the mandibles was retrieved for micro-computed tomography (CT) and methylene blue staining to analyze alveolar bone loss. The left side of the mandibles was histologically analyzed by TRAP and H&E staining. The MMP-9 mRNA level in gingival tissue was investigated by RT-PCR. Alveolar bone resorption was significantly reduced in the PerioH-035-treated groups. The number of dense multi-nucleated cells found to be TRAP-positive by staining in the ligatured rats was markedly decreased by PerioH-035 application. In addition, periodontal tissue destruction, especially cementum demineralization, was ameliorated in the P1 and P100 groups. Moreover, gingival tissue from the PerioH-035-treated group showed a decrease in the MMP-9 mRNA level, resulting in recovery of collagen degradation. These results suggest that PerioH-035 has therapeutic effects on periodontitis, and thus, PerioH-035 shows promise as a treatment for periodontitis.

Cytokine gene expression profiles during initiation, progression and resolution of periodontitis.

Variations in the expression of cytokines during the progression of periodontitis remain ill-defined. We evaluated the expression of 19 cytokine genes related to T-cell phenotype/function during initiation, progression and resolution of periodontitis, and related these to the expression of soft and bone tissue destruction genes (TDGs). A ligature-induced periodontitis model was used in rhesus monkeys (M.mulatta) (n=18). Gingival tissues were taken at baseline pre-ligation, 2weeks and 1month (Initiation) and 3months (progression) post ligation. Ligatures were removed and samples taken 2months later (resolution). Total RNA was isolated and the Rhesus Gene 1.0 ST (Affymetrix) used for gene expression analysis. Significant expression changes were validated by qRT-PCR. Disease initiation/progression was characterized by overexpression of Th17/Treg cytokine genes (IL-1β, IL-6, TGFβ and IL-21) and down-regulation of Th1/Th2 cytokine genes (IL-18 and IL-25). Increased IL-2 and decreased IL-10 levels were seen during disease resolution. Several Th17/Treg cytokine genes positively correlated with TDGs, whereas most Th1/Th2 genes exhibited a negative correlation. Initiation, progression and resolution of periodontitis involve over- and underexpression of cytokine genes related to various T-helper subsets. In addition, variations in individual T-helper response subset/genes during disease progression correlated with protective/destructive outcomes.

Porphyromonas gingivalis exacerbates ligature-induced, RANKL-dependent alveolar bone resorption via differential regulation of Toll-like receptor 2 (TLR2) and TLR4.

Toll-like receptors (TLRs) play a key role in the innate immune responses to periodontal pathogens in periodontal disease. The present study was performed to determine the roles of TLR2 and TLR4 signaling in alveolar bone resorption, using a Porphyromonas gingivalis-associated ligature-induced periodontitis model in mice. Wild-type (WT), Tlr2(-/-), and Tlr4(-/-) mice (8 to 10 weeks old) in the C57/BL6 background were used. Silk ligatures were applied to the maxillary second molars in the presence or absence of live P. gingivalis infection. Ligatures were removed from the second molars on day 14, and mice were kept for another 2 weeks before sacrifice for final analysis (day 28). On day 14, there were no differences in alveolar bone resorption and gingival RANKL expression between mice treated with ligation plus P. gingivalis infection and mice treated with ligation alone. Gingival interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) expression was increased, whereas IL-10 expression was decreased in WT and Tlr2(-/-) mice but not in Tlr4(-/-) mice. On day 28, WT and Tlr4(-/-) mice treated with ligation plus P. gingivalis infection showed significantly increased bone loss and gingival RANKL expression compared to those treated with ligation alone, whereas such an increase was diminished in Tlr2(-/-) mice. Gingival TNF-α upregulation and IL-10 downregulation were observed only in WT and Tlr4(-/-) mice, not in Tlr2(-/-) mice. In all mice, bone resorption induced by ligation plus P. gingivalis infection was antagonized by local anti-RANKL antibody administration. This study suggests that P. gingivalis exacerbates ligature-induced, RANKL-dependent periodontal bone resorption via differential regulation of TLR2 and TLR4 signaling.

Periodontitis-activated monocytes/macrophages cause aortic inflammation.

A relationship between periodontal disease and atherosclerosis has been suggested by epidemiological studies. Ligature-induced experimental periodontitis is an adequate model for clinical periodontitis, which starts from plaque accumulation, followed by inflammation in the periodontal tissue. Here we have demonstrated using a ligature-induced periodontitis model that periodontitis activates monocytes/macrophages, which subsequently circulate in the blood and adhere to vascular endothelial cells without altering the serum TNF-α concentration. Adherent monocytes/macrophages induced NF-κB activation and VCAM-1 expression in the endothelium and increased the expression of the TNF-α signaling cascade in the aorta. Peripheral blood-derived mononuclear cells from rats with experimental periodontitis showed enhanced adhesion and increased NF-κB/VCAM-1 in cultured vascular endothelial cells. Our results suggest that periodontitis triggers the initial pathogenesis of atherosclerosis, inflammation of the vasculature, through activating monocytes/macrophages.

Effect of Cimetidine on Nitro-Oxidative Stress in a Rat Model of Periodontitis.

Background and aimsPeriodontitis is a chronic inflammation that involves nitro-oxidative stress with damaging periodontal structural effects. We aimed to evaluate the consequences of low-dose cimetidine on nitro-oxidative stress in periodontitis.MethodsA rat model of ligature-induced periodontitis was used. After two weeks, the periodontitis groups were treated with cimetidine, aminoguanidine, N-nitro-L-arginine methyl ester and trolox for one week. On day 21, blood was drawn and the serum analyzed for measurement of total nitrites and nitrates, total oxidative status, total antioxidant response, and oxidative stress index.ResultsCimetidine had an inhibitory effect on the synthesis of nitric oxide (p=0.001), total oxidative status (p=0.01) and oxidative stress index (p=0.01). Total antioxidant reactivity was increased by cimetidine (p=0.01). The effects of cimetidine were almost like those of aminoguanidine, NG-nitro-L-arginine methyl ester, and trolox.ConclusionsLow-dose cimetidine can be used as adjunctive host modulatory therapy in chronic periodontitis because it reduces nitro-oxidative stress.

Optimization of the ligature-induced periodontitis model in mice

Periodontitis is a prevalent oral inflammatory disease that leads to alveolar bone loss and may exert an adverse impact on systemic health. Experimental animal models are critical tools to investigate mechanisms of periodontal pathogenesis and test new therapeutic approaches. The ligature-induced periodontitis model has been used frequently in relatively large animals, including non-human primates, to assess the host response and its effects on the tooth-supporting tissues (gingiva and bone) under well-controlled conditions. Although mice constitute the most convenient and versatile model for mechanistic immunological research (plethora of genetically engineered strains and immunological reagents), the tiny size of the murine oral cavity has presented technical challenges for ligature placement. In this report, we present a straightforward method for ligating the second maxillary molar tooth, and, moreover, identified the most appropriate sites for evaluating inflammatory bone loss in a valid and reproducible manner. These optimizations are expected to facilitate the use of the mouse ligature-induced periodontitis model and consequently contribute to better understanding of the immunopathological mechanisms of periodontitis.

Increased bone loss and amount of osteoclasts in kinin B1 receptor knockout mice

The pathophysiology of periodontal diseases involves aspects of immunity and bone remodelling. Considering the role of the kinin B1 receptor (Bdkrb1) in inflammation and healing, the purpose of this study was to evaluate the contribution of Bdkrb1 to the pathogenesis of periodontitis. We used a model of ligature-induced experimental periodontitis (LIEP) in mice lacking Bdkrb1 (Bdkrb1(-/-) ) to test the role of this receptor in bone loss and cytokine secretion by lymph nodes cells. Angiotensin-converting enzyme inhibitor (ACEi) was used as a pharmacological strategy to support the genetic model. Also, autonomous effect of Bdkrb1 deletion was evaluated in osteoclasts precursors from bone marrow. Bdkrb1(-/-) mice exhibit increased bone loss and IL-17 secretion in response to LIEP when compared to wild type. LIEP does not modify TNF-α, IFN-γ and IL-10 levels in Bdkrb1(-/-) mice after 21days. Bone marrow cells from Bdkrb1(-/-) displayed increased differentiation into functional osteoclasts with consistent artificial calcium phosphate degradation. Furthermore, treatment of mice with ACEi prevented bone destruction. Bdkrb1 participates in the pathogenesis of LIEP bone loss possibly through mechanisms that involve modulation of the TH 17 response, thereby demonstrating its role in the development of periodontitis.

Impact of Mucosal Inflammation on Oral Simian Immunodeficiency Virus Transmission

Mucosal tissues are the primary route of transmission for most respiratory and sexually transmitted diseases, including human immunodeficiency virus (HIV). There is epidemiological evidence that genital mucosal inflammation leads to enhanced HIV type 1 (HIV-1) transmission. The objective of this study was to assess the influence of periodontal inflammation on oral HIV transmission using a nonhuman primate model of teeth ligature-induced periodontitis. Simian immunodeficiency virus (SIV) was nontraumatically applied to the gingiva after moderate gingivitis was identified through clinical and immunologic analyses (presence of inflammatory cytokines). Overall oral SIV infection rates were similar in the gingivitis-induced and control groups (5 infections following 12 SIV administrations for each), although more macaques were infected with multiple viral variants in the gingivitis group. SIV infection also affected the levels of antiviral and inflammatory cytokines in the gingival crevicular fluid, and a synergistic effect was observed, with alpha interferon and interferon-inducible protein 10 undergoing significant elevations following SIV infection in macaques with gingivitis compared to controls. These increases in antiviral and inflammatory immune modulators in the SIV-infected gingivitis macaques could also be observed in blood plasma, although the effects at both compartments were generally restricted to the acute phase of the infection. In conclusion, while moderate gingivitis was not associated with increased susceptibility to oral SIV infection, it resulted in elevated levels of cytokines in the oral mucosa and plasma of the SIV-infected macaques. These findings suggest a synergy between mucosal inflammation and SIV infection, creating an immune milieu that impacts the early stages of the SIV infection with potential implications for long-term pathogenesis.

INOS‐Derived Nitric Oxide Stimulates Osteoclast Activity and Alveolar Bone Loss in Ligature‐Induced Periodontitis in Rats

Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long-term iNOS inhibition, and the differentiation and activity of OC from iNOS-knockout (KO) mice in vitro. Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild-type C57BL/6 mice. OC were counted 6 days later after tartrate-resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks. Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature-induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild-type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts. Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.

Effects of verbascoside, biotechnologically purified by Syringa vulgaris plant cell cultures, in a rodent model of periodontitis

Verbascoside has previously been characterized as an effective scavenger of active free radicals and an inhibitor of lipid peroxidation. In the present study, we have investigated the effects of verbascoside from Syringa vulgaris in a rat model of ligature-induced periodontitis. Male Sprague-Dawley rats were lightly anaesthetized with pentobarbitone (35 mg/kg). Sterile, 2-0 black braided silk thread was placed around the cervix of the lower left first molar and knotted medially. Animals received vebascoside 2 mg/kg orally, daily for 8 days. On the eighth day after placement of the ligature, we evaluated several markers of inflammation: (i) myeloperoxidase activity, (ii) thiobarbituric acid-reactant substance measurements, (iii) NF-κB expression, (iv) iNOS expression, (v) the nitration of tyrosine residues, (vi) activation of the nuclear enzyme poly(ADP-ribose) polymerase, (vii) Bax and Bcl-2 expression and (viii) a degree of gingivomucosal tissue injury. Oral administration of verbascoside (2 mg/kg daily for 8 days) significantly decreased all of the parameters of inflammation as described above. These results demonstrate that verbascoside exerts an anti-inflammatory role during experimental periodontitis and is able to ameliorate the tissue damage associated with ligature-induced periodontitis.

Evidence that metyrapone in the presence of inflammation modulates cytokine mRNA expression

Objective Metyrapone (MT) has been used clinically to decrease glucocorticoid levels in human and animal studies. However, the potential effects of MT in the presence of inflammation are poorly understood. Thus, the aim of this study was to evaluate the effects of the administration of MT on the mRNA levels of pro-inflammatory cytokines in the presence of inflammation induced by the well-established model of ligature-induced periodontitis in rats. Material and methods Sixty animals were randomly assigned into three experimental groups of 20 rats each: G1-control; G2-periodontal disease (PD) induced by cotton ligature; G3-PD associated with 3 daily doses of MT (50 mg/kg/3 × 3 h). After 30 days, all animals were killed by decapitation. Blood samples were taken and the concentrations of corticosterone and catecholamines measured. Marginal tissues around ligated and non-ligated teeth were harvested and gene expression was assessed by quantitative polymerase chain reaction technique (qPCR). Moreover, the area of interradicular bone loss (ABL) was histometrically determined. Results Data analysis showed that: (i) ligature placement resulted in a significant ABL, as compared to non-ligated sites of G1 group; (ii) mRNA levels of all the pro-inflammatory factors assessed (INF-γ, TNF-α, IL-1β and IL-6) were increased in the PD group (G2) ( p < 0.05) when compared to G1; (iii) there were no significant differences in corticosterone and catecholamine plasmatic levels between the three groups; (iv) MT administration, in the presence of inflammation, induces an increased ABL and significantly increased mRNA levels of all pro-inflammatory cytokines analyzed ( p < 0.05). Conclusion Within the limits of this study, it can be concluded that MT in the presence of inflammation may modulate expression of pro-inflammatory cytokines, regardless of its effect on plasma corticosterone levels.

Effect of Atorvastatin in radiographic density on alveolar bone loss in wistar rats

The purpose of this study was to evaluate the effect of Atorvastatin (ATV) on alveolar bone loss induced in rats. Periodontitis was induced by ligature placement around the upper second left molar in a total of 24 male Wistar rats (± 200 g). Groups of 6 animals received via oral gavage either saline or ATV (1, 3 and 9 mg/kg) during 11 days. After this time, the animals were sacrificed and their maxillae were removed, defleshed, radiographed by Digora System®, and latter stained to be photographed using a digital camera. Data were analyzed statistically by ANOVA and Bonferroni test at 5% significance level and presented as mean ± SEM. ATV (9 mg/kg) caused a significant increase on gray tone variation of over 48% (118.3 ± 12.0 gray tones) when compared to saline (79.8 ± 6.2 gray tones), indicating greater radiographic density. These data were corroborated by macroscopic findings, where ATV (9 mg/kg) reduced alveolar bone loss by over 47% (p<0.05), when compared to the group of untreated animals (saline). In summary, ATV was able to prevent alveolar bone loss seen on a ligature-induced periodontitis model.

WY-14643, a Potent Peroxisome Proliferator Activator Receptor-α PPAR-α Agonist Ameliorates the Inflammatory Process Associated to Experimental Periodontitis

We have investigated the effects of WY14643, a potent peroxisome proliferator activator receptor-α (PPAR-α) agonist, in a rat model of ligature-induced periodontitis. Male Sprague-Dawley rats were lightly anaesthetized with pentobarbitone (35 mg/kg). Sterile, 2-0 black braided silk thread was placed around the cervix of the lower left first molar and knotted medially. Animals received WY14643 (1 mg/kg i.p, daily for eight days). Eighths days after placement of the ligature, we evaluated several markers of inflammation such us (1) myeloperoxidase activity, (2) a cytokines and adhesion molecules expression, (3) NF-κB expression, (4) iNOS expression, (5) the nitration of tyrosine residues, (6) activation of the nuclear enzyme poly(ADP-ribose) polymerase, (7) apoptosis, and (8) the degree of gingivomucosal tissues injury. Administration of WY14643 significantly decreased all of the parameters of inflammation as described above. These results demonstrate that WY14643 exerts an anti-inflammatory role during experimental periodontitis and is able to ameliorate the tissue damage.