Signs of alveolar bone damage in early stages of periodontitis and its prevention by stimulation of cannabinoid receptor 2. Model in rats / Signos de daño óseo alveolar en estadios tempranos de periodontitis y su prevención por estimulación del receptor cannabinoide 2. Modelo en ratas microcisalhamento do cimento resinoso

ABSTRACT The aims of the present study were, first, to identify signs of alveolar bone damage in early stages of experimental periodontitis (EP) and, second, to assess its possible prevention by treatment with cannabinoid receptor 2 agonist HU 308. Experimental periodontitis was induced by injections of lipopolysaccharide (LPS) (1mg/ml) in gums surrounding maxillary and mandibular first molar, 3 days per week, and untreated controls were kept for comparison. Then, a 3-week study was conducted including eighteen new rats (six rats per group): 1) controls; 2) experimental periodontitis rats; and 3) experimental periodontitis rats treated daily with HU 308 (500 ng/ml). After euthanasia, alveolar bone loss was assessed by morphometric and histomorphometric techniques, and the content of prostaglandin E2 (PGE2) in gingival tissue was evaluated by radioimmunoassay. The first signs of alveolar bone loss were apparent at 3 weeks of experimental periodontitis (ρ<0.05) in the mandibular first molar, but there was no detectable change at 1 week, leading us to establish 3 weeks as an early stage of experimental periodontitis. Rats subjected to 3-week experimental periodontitis showed less interradicular bone volume, less whole bone perimeter and fewer bone formation areas, and higher periodontal space height, bone resorption areas, number of osteoclasts and gingival content of prostaglandin E2 than controls, while HU 308 prevented, at least partially, the deleterious effects (ρ<0.001). We can conclude that a 3-week term of lipopolysaccharide-induced periodontitis in rats provides a valid model of the early stage of the disease, as emerging damage is observed in bone tissue. Furthermore, harmful effects at 3 weeks could be prevented by local stimulation of cannabinoid receptor 2, before greater damage is produced.

RESUMEN El objetivo del presente trabajo fue, en primer lugar, identificar signos de daño óseo alveolar en estadios tempranos de periodontitis experimental y, en segundo lugar, evaluar su posible prevención mediante el tratamiento con el agonista del receptor cannabinoide 2, HU 308. La periodontitis experimental fue inducida por inyecciones de lipopolisacárido (1mg/ml) en la encía circundante al primer molar maxilar y mandibular, 3 días por semana, en tanto que controles no tratados fueron mantenidos para la comparación. Posteriormente, un estudio de 3 semanas con dieciocho nuevas ratas (seis por grupo) fue desarrollado: 1) controles; 2) ratas con periodontitis experimental, y 3) ratas con periodontitis experimental tratadas diariamente con HU 308 (500ng/ml). Luego de la euthanasia, la pérdida ósea alveolar fue evaluada por técnicas morfométricas e histomorfométricas, y el contenido de prostaglandina E2 en el tejido gingival fue determinado por radioinmunoensayo. Los primeros signos de pérdida ósea alveolar fueron evidentes a las 3 semanas de inducción de periodontitis experimental (ρ<0.05) en el primer molar mandibular, mientras que no hubo cambios detectables luego de 1 semana de inducción, hecho que nos condujo a establecer a las 3 semanas como un estadio temprano de periodontitis experimental, Las ratas sometidas a perdiodontitis experimental de 3 semanas mostraron menor volumen óseo interradicular, menor perímetro óseo y menos áreas de formación ósea, y mayor altura del espacio periodontal, más áreas de reabsorción ósea, mayor número de osteoclastos y mayor contenido gingival de prostaglandina E2, en comparación a los controles, mientras que el tratamiento con HU 308 previno, al menos parcialmente, los efectos deletéreos (ρ<0.001). Podemos concluir que el término de 3 semanas de periodontitis inducida por lipopolisacárido es un modelo válido de estadio inicial de la enfermedad experimental, dado que se evidencia daño emergente en el tejido óseo. Asimismo, los efectos deletéreos de 3 semanas podrían ser prevenidos por la estimulación local del receptor cannabinoide 2, antes que un daño mayor sea producido.

Alveolar bone pattern and salivary leptin levels among premenopausal obese women / Padrão ósseo alveolar e níveis salivares de leptina entre mulheres obesas na pré-menopausa

ABSTRACT Background: Systemic bone loss may lead to more severe periodontal destruction, decreasing local bone mineral density. Aim: A cross-sectional designed was performed to study associations among alveolar bone pattern, salivary leptin concentrations, and clinical periodontal status in premenopausal obese and eutrophic women. Methods: Thirty morbid obese (G1) and 30 normal-weight (G2) women were included. Anthropometric and periodontal measurements (probing depth - PD, clinical attachment levels - CAL, presence of calculus, bleeding on probing -BOP, and plaque accumulation) were assessed. OHIP-14 was used for assessment of oral health impact on quality of life. Panoramic radiography was used to obtain the panoramic mandibular index (PMI), mandibular cortical index (MCI), and mental index (MI). Intraoral periapical (PA) radiography was taken to measure the total trabecular bone volume. Leptin was measured in saliva of fasted overnight women. Results: Groups 1 and 2 differed in all anthropometric aspects, but height. Pocket depth, calculus, BOP, and plaque index were worse in G1. No differences between groups were found considering OHIP. Normal-weight subjects showed higher proportion of dense bone trabeculae than obese subjects for pre-molars, but not for molars. Mental and panoramic mandibular indexes did not differ and were in normal level. Leptin concentration was dependent only on BMI. Conclusion: Obesity affected the periodontal conditions, the alveolar bone pattern, and the salivary leptin concentration.

RESUMO Racional: A perda óssea sistêmica pode levar à destruição periodontal mais severa, diminuindo a densidade mineral óssea local. Objetivo: Investigar as associações entre padrão ósseo alveolar, concentrações de leptina salivar e estado periodontal em mulheres obesas na pré-menopausa e eutróficas. Métodos: Foram avaliadas 30 mulheres com obesidade mórbida (G1) e 30 com peso normal (G2). Foram analisadas as medidas antropométricas e periodontais (profundidade de sondagem - PS, nível clínico de inserção - NCI, presença de cálculo, sangramento à sondagem - SS e acúmulo de placa). O impacto da saúde bucal na qualidade de vida foi mensurado por meio do questionário OHIP-14. Radiografia panorâmica foi utilizada para obter o índice mandibular panorâmico (PMI), índice cortical mandibular (MCI) e índice mental (MI); já a radiografia periapical intraoral (AF) para medir o volume ósseo trabecular total. A leptina salivar foi coletada no período da manhã com a paciente em jejum. Resultados: Os grupos 1 e 2 diferiram em todos os aspectos antropométricos, exceto em estatura. Profundidade de bolsa, cálculo, SS e índice de placa foram piores no G1. Não foram encontradas diferenças entre os grupos considerando o OHIP. Indivíduos com peso normal apresentaram maior proporção de trabéculas ósseas densas do que os obesos para pré-molares, mas não para molares. Índices radiomorfométricos não diferiram entre os grupos e estavam dentro de valores normais. A concentração de leptina esteve associada ao IMC. Conclusão: A obesidade afetou as condições periodontais, o padrão ósseo alveolar e a concentração de leptina salivar.

The role of potassium channels in the endothelial dysfunction induced by periodontitis

Abstract Objective: Periodontitis is associated with endothelial dysfunction, which is clinically characterized by a reduction in endothelium-dependent relaxation. However, we have previously shown that impairment in endothelium-dependent relaxation is transient. Therefore, we evaluated which mediators are involved in endothelium-dependent relaxation recovery. Material and methods: Rats were subjected to ligature-induced experimental periodontitis. Twenty-one days after the procedure, the animals were prepared for blood pressure recording, and the responses to acetylcholine or sodium nitroprusside were obtained before and 30 minutes after injection of a nitric oxide synthase inhibitor (L-NAME), cyclooxygenase inhibitor (Indomethacin, SC-550 and NS- 398), or calcium-dependent potassium channel blockers (apamin plus TRAM- 34). The maxilla and mandible were removed for bone loss analysis. Blood and gingivae were obtained for C-reactive protein (CRP) and myeloperoxidase (MPO) measurement, respectively. Results: Experimental periodontitis induces bone loss and an increase in the gingival MPO and plasmatic CRP. Periodontitis also reduced endothelium-dependent vasodilation, a hallmark of endothelial dysfunction, 14 days after the procedure. However, the response was restored at day 21. We found that endothelium-dependent vasodilation at day 21 in ligature animals was mediated, at least in part, by the activation of endothelial calcium-activated potassium channels. Conclusions: Periodontitis induces impairment in endothelial-dependent relaxation; this impairment recovers, even in the presence of periodontitis. The recovery is mediated by the activation of endothelial calcium-activated potassium channels in ligature animals. Although important for maintenance of vascular homeostasis, this effect could mask the lack of NO, which has other beneficial properties.

Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats

Abstract This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

Effect of 15% Alcohol Dependence on Alveolar Bone Loss and TNF-α Secretion in Wistar Rats

Abstract The aim of the present study was to evaluate the effect of 15% alcohol dependence on ligature-induced alveolar bone loss and TNF- secretion in Wistar rats. Thirty-three male Wistar rats aged 45-60 days (mean weight=253 g) were randomly allocated test or control groups. Test group (n=18) received 15% alcohol as liquid intake and control group (n=15) received water during the experimental period. TNF-α was analyzed by ELISA assay in 11 animals per group. After 14 days of alcohol/water intake, alcohol dependency was assessed and silk ligatures were placed around the left second upper molars. Ligature presence and body weight were checked weekly. After 40 days, animals were sacrificed and the maxillae were defleshed for morphometric analysis using standardized images. All animals in the test group displayed signs of alcohol dependency at day 14. No statistically significant differences in final body weight (334.83±21.38 vs. 322.48±30.65 g, p=0.20) were observed between groups. In relation to alveolar bone loss, no statistically significant difference was observed among test and control groups both for ligated teeth (0.76±0.06 vs. 0.74±0.10 mm, p=0.60) and unligated teeth (0.41±0.16 vs. 0.35±0.05 mm, p=0.22). The TNF-α secretion also did not display statistically significant differences between test and control groups (10.78±1.84 vs. 12.13±2.11 pg/mL, p=0.12). It may be concluded that 15% alcohol dependency was not capable to alter alveolar bone loss and TNF-α secretion in Wistar rats.

Resumo O objetivo do presente estudo foi avaliar o efeito da dependência de álcool a 15% sobre a perda óssea alveolar induzida e secreção de TNF-α em ratos Wistar. Trinta e três ratos wistar com idade entre 45 e 60 dias (peso médio=253 g) foram alocados aleatoriamente para o grupo teste ou controle. O grupo teste (n=18) recebeu álcool a 15% como ingestão líquida e o grupo controle (n=15) recebeu água durante o período experimental. TNF-α foi analisado por meio de ELISA em 11 animais por grupo. Após 14 dias de ingestão de álcool/água a dependência do álcool foi determinada e ligaduras de seda foram colocadas ao redor dos segundos molares superiores esquerdos. A presença das ligaduras e o peso corporal foram verificadas semanalmente. Depois de 40 dias os animais foram sacrificados e as maxilas foram preparadas para análise morfométrica em fotografias estandardizadas. Todos os animais do grupo teste apresentaram sinais de dependência de álcool no dia 14. Não foram observadas diferenças estatisticamente significativas no peso corporal final entre os grupos (334,83±21,38 vs. 322,48±30,65 gramas, p=0,20) Em relação a perda óssea alveolar, não foram observadas diferenças estatisticamente significativas entre os grupos teste e controle tanto para dentes com (0,76±0,06 vs. 0,74±0,10 mm, p=0,60) como para dentes sem ligadura (0,41±0,16 vs. 0,35±0,05 mm, p=0,22). A secreção de TNF-α também não demonstrou diferenças estatisticamente significativas entre os grupos teste e controle (10,78±1,84 vs. 12,13±2,11 pg/mL, p=0,12). Pode-se concluir que a dependência de álcool a 15% não foi capaz de alterar a perda óssea alveolar e a secreção de TNF-α em ratos Wistar.

O sistema renina-angiotensina na doença periodontal induzida experimentalmente em ratos / The renin-angiotensin system in experimentally-induced periodontal disease in rats

A doença periodontal (DP) compreende um grupo de lesões que afetam os tecidos periodontais de proteção (gengivite) e suporte (periodontite), envolvendo a participação de células residentes, células estruturais e mediadores inflamatórios. Pesquisa recente do nosso laboratório mostrou a existência de um Sistema Renina-Angiotensina (SRA) local no tecido gengival de ratos e sugeriu que o SRA possa ter participação na DP. Portanto, o objetivo deste trabalho foi avaliar a se o SRA está envolvido na iniciação e na progressão da DP induzida experimentalmente em ratos. Para tanto, foi utilizado modelo de indução da DP por colocação de ligadura, por 7 e 14 dias, ao redor do primeiro molar inferior de ratos e tratamento destes animais com drogas que afetam o SRA [losartan (50 mg/Kg/dia), alisquireno (30 mg/Kg/dia) ou enalapril (10 mg/Kg/dia)]. Foram realizadas técnicas de análise da perda óssea alveolar, reação em cadeia da polimerase (PCR) quantitativa e imunoistoquímica. Após a coleta, os dados foram devidamente analisados por meio de gráficos e tabelas, sendo utilizada ANOVA a 2 e 3 critérios e adotado nível de significância de 5%. Em nível protéico, houve aumento significativo da maioria dos componentes do SRA (p<0,05) na DP. A renina apresentou aumento nos tratamentos com losartan, alisquireno e enalapril tanto nos animais sham (cirurgia fictícia de indução da DP) quanto nos animais com DP, aos 7 e 14 dias, e não apresentou marcação no grupo controle (água), demonstrando efeito dependente dos tratamentos farmacológicos. Na DP houve aumento dos componentes AT1 (aos 7 e 14 dias), AT2 (aos 7 dias) e enzima conversora da angiotensina (ECA; aos 7 e 14 dias) nos grupos tratados com losartan, alisquireno e enalapril. Também houve aumento de imunomarcação nos animais com DP para AT2 (aos 14 dias) e ECA (aos 14 dias) em animais do grupo controle. Em relação à expressão gênica, houve aumento da expressão de RNAm nos animais com DP para o...

Periodontal disease (PD) comprises a group of lesions that affect protection (gingivitis) and support periodontal tissues (periodontitis) involving the participation of resident and structural cells as well as inflammatory mediators. Recent research in our laboratory showed the existence of a local gingival renin-angiotensin system (RAS), and suggested that it might participate in PD. Therefore, the aim of this study was to evaluate whether the RAS is involved in the initiation and progression of the experimentally-induced PD in rats. For this purpose, a model of ligature placement, for 7 and 14 days, around the lower first molar in rats, and the treatment of such animals with drugs that affect the RAS [losartan (50 mg/Kg/day), aliskiren (30 mg/Kg/day) or enalapril (10 mg/Kg/day)] were employed. The following techniques were performed: alveolar bone loss analysis, quantitative real-time polymerase chain reaction and immunohistochemistry. Data were collected, organized in tables and graphs, and submitted to 2 and 3 way ANOVA with significance level established at 5%. In the protein level, there was a significant increase in the majority of the RAS components in PD. Immunolocalization for renin increased when animals were treated with losartan, aliskiren or enalapril, for 7 and 14 days, in both sham (fictitious surgery for PD induction) and PD animals, whereas the control group (water) had no staining, demonstrating a drug-related effect. In animals with PD treated with losartan, aliskiren or enalapril there was an increase in staining for AT1 (at 7 and 14 days), AT2 (at 7 days) and angiotensin-converting enzyme (ACE; at 7 and 14 days). There was also increased staining in PD animals for AT2 (at 14 days) and ACE (at 14 days) in the control group. As far as genic expression, there was an increase in mRNA expression for AT2 in control animals with PD (at 7 and 14 days), and in the animals treated with losartan or enalapril (at 7 days)...

Detection and measurement of oral malodour in periodontitis patients.

BACKGROUND & OBJECTIVES: Malodour has been correlated with the concentration of volatile sulphur compounds produced in the oral cavity by metabolic activity of bacteria colonizing the periodontal sites and the dorsum of the tongue. The aim of this study was to detect malodour in mouth air organoleptically and using a portable sulphide monitor and to correlate it with the clinical parameters, halitosis linked toxins and BANA, using tongue and subgingival plaque samples. The halitosis grading is also correlated with the microbial colonies of the subgingival plaque sample. METHODS: 20 patients with chronic periodontitis with 5-7 mm pocket depth, radiographic evidence of bone loss and presence of oral malodour participated in this study. Assessment of mouth air was done organoleptically and by using a portable sulphide monitor. The clinical parameter, plaque index (PI), gingival index (GI), gingival bleeding index (BI), were obtained from all the areas. Samples for BANA and to detect halitosis linked toxins were taken from the dorsal surface of the tongue and periodontal pockets ranging 5-7 mm. Halitosis related microbial colonies were identified using anaerobic culturing from the subgingival plaque. RESULTS: The scores of PI, GI, BI and sample that tested positive for halitosis linked toxins and with the halitosis grading were not significant. The presence of tongue coating and the halitosis grading and toxin levels were significant. BANA has shown to be non contributory due to technical problems. Anaerobic culture has shown to identify Streptococcus, Bacteroides, Fusobacterium, Porphyromonas and Prevotella colonies. INTERPRETATION & CONCLUSION: The results confirmed that there was no correlation between the clinical parameters, halitosis linked toxins and halitosis grading. The microbial colonies have shown to correlate with the presence of oral malodour.

Determination of systemically & locally induced periodontal defects in rats.

BACKGROUND & OBJECTIVE: The role of lathyrogens on bone metabolism is unclear, therefore we undertook this study to observe periodontal and systemic alterations in experimental lathyrism in rat and compare these changes to that observed in the locally induced periodontitis group. METHODS: A total of 45 male Wistar rats were equally divided in the lathyritic group (group 1), ligature-induced periodontitis group (group 2), and healthy controls (group 3). Experimental lathyrism was induced by once daily subcutaneous administration of beta-aminoproprionitrile (beta-APN), at a dose of 5 mg/0.4 ml per 100 g of body weight for 40 days. Ligature-induced periodontitis was created by tying silk ligatures on the necks of mandibular molars. After 40 days, blood samples were obtained and the animals were decapitated. Radiographic observations, extraction tests, histologic evaluations were performed, and serum ALP activity and gingival tissue IL-1beta levels were measured. RESULTS: Significant alveolar bone resorption around the mandibular molar teeth (P<0.001); lower extraction force levels (P<0.001); higher numbers of lymphocytes and macrophages (P<0.01) (both in connective tissue and epithelium at the dentogingival junction); decreased ALP activity (P<0.001); and increased gingival tissue IL-1beta levels (P<0.001) were observed in groups 1 and 2, compared to those in group 3. ALP activity was higher in group 1 than in group 2 rats (P<0.05). INTERPRETATION & CONCLUSION: Similar radiographical and histopathological findings and comparable increases in gingival tissue IL-1beta levels both in groups 1 and 2 showed that in addition to resorption of alveolar bone, chronic inflammation of periodontium also occurred both in the lathyritic rats as well as in ligature-induced periodontitis group rats.