Material and methods

Forty 10-week-old male C57BL6/J mice were used in this experiment. They were randomly divided into 2 groups: (1) treated with tap water (VH) as the control group, and (2) treated with 10 mg per kilogram per day of LOS (Sigma-Aldrich, St Louis, Mo). The sample size calculation was based on a previous study from our group. No significant weight loss was observed. All treatments followed the ethical regulations of the institutional ethics committee of the Pontifıcal Catholic University of Minas Gerais.

Daily gavages of 100 μL of 10 mg per kilogram per day of LOS or tap water were administered to the LOS and VH groups, respectively. The drug was administered for 6 or 12 days for the histopathologic assays and for 0 or 12 hours for the molecular analyses.

Tooth movement was induced as previously described. Briefly, the mice were anesthetized with 0.2 mL of a xylazine (0.02 mg/mL) and ketamine (50 mg/mL) solution. An orthodontic appliance consisting of a nickel-titanium 0.25 × 0.76-mm coil spring (Lancer Orthodontics, San Marcos, Calif) was bonded by light-cured resin (Transbond; 3M Unitek, Monrovia, Calif) between the maxillary right first molar and both maxillary incisors ( Fig 1 ). The magnitude of force was calibrated by a tension gauge (Shimpo Instruments, Itasca, Ill) to exert a force of 35 g in the mesial direction. No reactivation was performed during the entire experimental period. For the histopathologic analysis, the left side of the maxilla (without an orthodontic appliance) was used as the control.

A, Occlusal view of the maxillae before placing the orthodontic device; B, a nickel-titanium open-coil spring was placed between the maxillary right first molar and incisor.

The mice were killed after 6 or 12 days for the histopathologic analysis or after 0 or 12 hours for the molecular analysis. For each set of experiments, 5 animals were used at each time point.

As was previously described, the right and left maxillae halves were fixed in 10% buffered formalin (pH 7.4), decalcified in 14% ethylenediaminetetraacetic acid (pH 7.4) for 20 days, and embedded in paraffin. The samples were cut into sagittal sections 5 μm thick. The sections were stained with tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich), counterstained with hematoxylin, and used for the histologic examinations. The mesial periodontal site of the distobuccal root of the first molar was used for osteoclast counts on 5 sections per animal. Osteoclasts were identified as TRAP-positive, multinucleated cells on the bone surface.

Measurement of OTM was performed as previously described. Images of the first and second molars were obtained using an optical microscope (Axioskop 40; Carl Zeiss, Gottingen, Germany) and an adapted digital camera (PowerShot A620; Canon, Tokyo, Japan). ImageJ software (National Institutes of Health) was used to quantify the degree of OTM by measuring the distance between the cementoenamel junction of the first and second molars on the right hemimaxilla in relation to the same measurements for the left hemimaxilla. Five vertical sections per animal were evaluated, and 3 measurements were made for each evaluation; the variability was less than 5%.

RNA extraction and real-time polymerase chain reaction were performed for each animal separately, as previously described. The periodontal ligament and the surrounding alveolar bone were extracted from the maxillary first molars with a stereomicroscope. All gingival tissues, oral mucosa, and teeth were discarded. The periodontal tissues and the alveolar bone that were extracted from the distal area of the distobuccal root of the maxillary first molar were considered the tension site. The mesial area of the same root was considered the compression site. The bone samples (4-8 mg) were frozen in liquid nitrogen after collection and then stored at –80°C. Previous to mRNA extraction, each sample was mechanically fragmented in liquid nitrogen using a bone chisel and homogenized in sterile Milli-Q water with Ultra Turrax (IKA Laboratory Equipment, Staufen, Germany), and subsequently submitted to RNA extraction using TRIZOL reagent (Life Technologies, Carlsbad, Calif), following the manufacturer’s instructions. The mRNA quality and RNA integrity number were assessed with the BioAnalyzer (Agilent, Santa Clara, Calif). The RNA integrity number results ranged between 7.25 and 8.66 (mean, 7.87; SD, 0.36).

Complementary DNA was synthesized using 2 μg of RNA through a reverse transcription reaction (Superscript II; Life Technologies). The targets included molecules known to regulate osteoclast functions: RANK (receptor activator of nuclear factor kappa-B), RANKL, osteoprotegerin, cathepsin K, and metalloproteinase 13. We also analyzed the expression of molecules known to regulate osteoblast functions: periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, and metalloproteinase 2. The analyses were performed in ViiA7 equipment (Applied Biosystems, Warrington, United Kingdom) with real-time polymerase chain reaction using TaqMan chemistry (Life Technologies) with inventoried optimized primers/probes sets (Life Technologies) as follows: molecules known to regulate osteoclast functions: RANK (catalog number 433118), RANKL (catalog number 4331182), osteoprotegerin (catalog number 433118), cathepsin K (catalog number 4331182), and metalloproteinase 13 (catalog number 4331182); and molecules known to regulate osteoblast functions: periostin (catalog number 4331182), dentin matrix protein (catalog number 4331182), alkaline phosphatase (catalog number 4331182), collagen 1A1 (catalog number 4331182), semaphorin 3A3 (catalog number 4331182), and metalloproteinase 2 (catalog number 4331182). The basic reaction conditions (40 cycles) were 95°C (10 minutes), 94°C (1 minute), 56°C (1 minute), and 72°C (2 minutes). The mean cycle threshold (Ct) values from duplicate measurements were used to calculate the expression of the target gene, with normalization to an internal control (β-actin) using the 2 ^−ΔΔCt formula.

Some researchers have used a dosage of antihypertensive drugs that promotes a reduction in blood pressure by 27%. In our study, we chose to use a dose to keep the blood pressure stable to eliminate bias. The mean arterial pressure measurement was evaluated by the tail-cuff method, which is a noninvasive computerized system for measuring blood pressure (Kent Scientific, Torrington, Conn). This tail-cuff blood pressure system uses volume-pressure recording technology to measure the blood pressure in the mouse’s tail. The animals were acclimated to the restraint and tail-cuff inflation for 1 day before the experiments. The restraint platform was maintained at approximately 32°C to 34°C. For each section, the mouse was placed in an acrylic box restraint, and the tail was inserted into a compression cuff that measured the blood pressure 15 times. The average of these values was calculated ( Table ).