Overview of the experimental design for induction of periodontitis by ligature (Experimental design 1).

<p>Mice received daily intraperitoneal injections of PBS, CSC or nicotine for 3 consecutive days (days −3 to −1). Mice were examined on day 7 after placement of the ligature.</p

Schematic illustration of the measurement of the distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC).

<p>Distance from the CEJ to the ABC was measured at four points indicated on the first molar to the third molar.</p

Overview of experimental design for alveolar bone repair after ligature removal (Experimental design 2).

<p>Mice were given daily intraperitoneal injections of PBS, CSC or nicotine for 3 consecutive days (days −3 to −1). Some of the mice were examined on day 7 after placement of the ligature, and some of the mice were examined 10 days after ligature removal. Three mice with nicotine-treated were given additional intraperitoneal injection of nicotine for 3 consecutive days (days 8–10) and examined 10 days after ligature removal.</p

Micro-CT analysis.

<p>(A) The maxillae were scanned by micro-computed tomography. (B) Measurement of total bone loss. Bar graphs represent the mean values ± SD (n = 3). * <i>p</i> < 0.01, vs. without ligation. ** <i>p</i> < 0.01, vs. PBS-treated. Dashed line means baseline (unligated side in PBS treatment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155594#pone.0155594.g003" target="_blank">Fig 3B</a>).</p

TRAP staining.

<p>(A) Detection of osteoclasts in tissue sections by tartrate-resistance acid phosphate (TRAP) staining. (B) Quantification of TRAP-positive osteoclasts. The mean of number of positive cells was determined from five serial sections per mouse. Data are represented as mean ± SD (n = 7). * <i>p</i> < 0.01, vs. without ligation. ** <i>p</i> < 0.01, vs. PBS-treated.</p

RANKL expression in submandibular lymph nodes.

<p><i>Rankl</i> mRNA expression in submandibular lymph nodes was assessed by real-time PCR. Quantitative mRNA values were normalized to the amount of <i>Gapdh</i>. Bar graphs represent the mean values ± SD (n = 7). * <i>p</i> < 0.01, vs. without ligation. ** <i>p</i> < 0.01, vs. PBS treated. Lower panels are images of submandibular lymph nodes. Representative lymph nodes are shown.</p

Histological analysis.

<p>Histological analysis of alveolar bone between the first molar and the second molar with or without a ligature. Decalcified periodontal tissue sections were prepared and stained with hematoxylin and eosin solution. Representative tissue sections are shown.</p