Após o período de 15 dias da indução da periodontite, os animais foram medicados diariamente via oral por gavagem durante 30 dias de acordo com o grupo de tratamento: o grupo alendronato (ALN) que recebeu a dose de 2 mg/kg de alendronato; o grupo resveratrol (RESV) que recebeu a dose 10 mg/kg de resveratrol dissolvido em 10 mg/ml de amido solúvel; grupo β- ciclodextrina/ resveratrol (β- CD/RESV) que recebeu a dose 10 mg/kg em uma proporção de 1: 1 Mol de β- ciclodextrina e o grupo controle que recebeu a dose de 10 mg/kg de β- CD.
33 A forma de administração dos medicamentos foi via oral através de uma cânula de gavagem (agulha de aço inox curvo 22 GA- Ciencor, com diâmetro de 1,0 mm, esfera de 1,7 mm e comprimento de 31 mm) 1 vez ao dia durante 30 dias.
Ao término do período de tratamento os animais foram eutanasiados.
1.7. Eutanásia
Após 30 dias de tratamento, os ratos foram eutanasiados pela inalação de dose excessiva de isoflurano. Sequencialmente as maxilas foram separadas em direita e esquerda e armazenadas em solução de formaldeído a 10% em tampão fosfato de sódio (Sigma-Aldrich ®, Saint Louis, MO, EUA).
Figura 4 - demonstração da aplicação da gavagem intragástrica
Figura 5 - demonstração da aplicação da gavagem intragástrica
34
1.8. Tomada radiográfica
Para quantificar a perda óssea ocasionada pela periodontite após o tratamento com alendronato e resveratrol, as hemimaxilas foram radiografadas utilizando um raio-x digital (X DENT Equipamentos Odontológicos LTDA) com um sensor intraoral digital 3x4 (NewIda), expostas aos seguintes parâmetros: 70 Kvp, 7 mA, distância foco-filme de 30 cm e tempo de exposição de 0,17s, com o feixe do raio-x posicionado perpendicularmente às peças em posicionador.
1.9. Calibração do examinador
Para assegurar a reprodutibilidade das medidas radiográficas, um único examinador foi calibrado realizando cada análise de medida com repetição de três vezes dentro de um período de 7 dias com coeficiente de correlação intraclasse de 0,99 - CCI 95 %.
1.10. Análise radiográfica
A análise das imagens foi realizada sob uma mesma luz artificial em um computador Samsung Expert X23 com monitor de 15,6 polegadas, com o auxílio do software ImageJ ® (https://ImageJ.nih.gov/ij/). Antes de realizar as medidas no programa, foi executado o escalonamento utilizando como parâmetro uma régua micrométrica de 1 mm.
Cada imagem foi ampliada duas vezes no programa. As análises foram realizadas entre as proximais dos primeiros e segundos molares superiores. Para padronizar as medidas, foi traçada uma linha entre a junção esmalte cemento do primeiro ao segundo molar superior. A partir desta linha foram demarcadas outras três linhas,
35 perpendiculares à primeira, até a crista óssea alveolar da região interproximal dos molares. Os valores das três linhas foram tomados em milímetros e transformados em uma única média.
Figura 6 - Desenho esquemático representando a metodologia de medida da reabsorção óssea realizada da JEC-COA. Linha (tracejada) entre as JEC do primeiro e segundo molar superior. Linha do ponto mais alto da COA até JEC em três posições distintas
1.11. Análise estatística
A normalidade de distribuição das variáveis foi avaliada atrás do teste Kolmogorov- Smirnov. As médias de todas as medidas radiográficas foram submetidas ao programa IMB SPSS Statistics (SPSS 22.0, SPSS Inc., Chicago, Illinois, USA), onde foi feito o teste de Kruskal Wallis e Mann-Whitney para comparação entre os grupos. O nível de significância estabelecido foi de 0,05.
36
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