20 The allocation of rats was performed at random also to allow for equal chances of being included in each group. Additionally, measurements in the morphometric analysis were performed by a calibrated and blinded examiner. The intra-class correlation coefficient of the examiner warrants excellent levels of reproducibility. Moreover, recent studies indicate that alveolar bone loss in mice and rats can be accurately quantified using microscopic evaluation, morphometric analysis, or microcomputed tomography with no significant variations in outcomes.17 and 21 To evaluate general health, rats were weighted weekly throughout the study. It is possible to infer that the general health remained

good, since no statistically significant differences were found amongst Panobinostat groups during the experimental period. Ligature-induced periodontal breakdown and this model are widely used in the literature.11, 14, PS-341 molecular weight 16 and 22 The experimental period of ligature in the literature varies from 15 to 60 days.

The period used in our study was based in data from a previous report, which showed that the main intra-group differences were found in the first 15 days.23 After this period, the differences levelled off. Additionally, similar pattern of bone loss was described in studies that used the same methodology and an experimental period of approximately 30 days.10, 12 and 14 The observed statistically significant

differences amongst teeth with and without ligatures support that the period of 15 days was sufficient. In relation to the corticosteroid administration (inhalation), Elias et al.15 verified that a forced ventilation chamber is an effective way for inhalation of steroidal anti-inflammatory drugs in rats. Despite the fact that histometric analysis has been used to measure bone loss in rats, morphometric analysis is an important tool validated in literature and today is used more frequently.11, 17 and 21 It can be an alternative to histometric analysis if the study aims to measure the bone loss. This is related to the ease of the technique and also to the fact that the direct view of defects can be made in this type of analysis without interference from Montelukast Sodium the cutting plans of histological sections.24 The concentrations of the medication used in the present study were chosen in accordance with previous studies.15, 25 and 26 The dose of 30 μg/ml was the concentration that caused behaviour alterations in animals, and 100 μg/ml was the lowest therapeutic dose used for asthma treatment in humans. We analysed different concentrations of the medication to verify a possible dose–response relationship. However, the results showed no dose–response relationship in this study. In the present study, a different pattern of alveolar bone loss amongst experimental groups was not detected.