Fifty-two six-week-old male Wistar rats were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). Rats were maintained under specific pathogen-free conditions in the Osaka Dental University Animal Care facility. The use of animals and experimental protocol were approved by the Committee for Animal Experiments of Osaka Dental University (#17-02018). The experimental procedures were executed in accordance with the Guidelines for Animal Experiments of Osaka Dental University.

Forty-six rats at seven weeks of age were anesthetized by inhalation of isoflurane (Wako Pure Chemical industries, Osaka, Japan) and intraperitoneal injection of pentobarbital (0.3 mg/kg) (Kyoritsu Seiyaku Corporation, Tokyo, Japan). Periodontitis was induced by intragingival injection of LPS from Porphyromonas gingivalis (LPS-PG) (InvivoGen, San Diego, CA, USA). LPS-PG was dissolved in endotoxin-free water at concentrations of 0.01, 0.1, and 1 mg/ml, and 5 μl of LPS-PG (for controls 5 μl of endotoxin-free water) was injected into the palatal gingiva around the second maxillary molars using a 31-gauge needle equipped with a gastight syringe (Hamilton Company, Reno, NV, USA) on day 1, day 3, and day 5.

For micro-CT analysis, 5 μl of 1 mg/ml LPS-PG was injected into the palatal gingiva around the second maxillary molars in three rats on day 1, day 3, and day 5, and rats were euthanized with an overdose of isoflurane and pentobarbital on day 14. Maxillary bones were excised and analyzed using a micro-CT scanner, SMX-130CT (Shimadzu Corporation, Kyoto, Japan) as described previously [21] . Three dimensional images were created using VGStudio MAX 1.2.1 software (Volume Graphics, GmbH, Germany).

For RNA extraction, rats were euthanized with an overdose of isoflurane and pentobarbital on day 9. Palatal gingiva around the second maxillary molars were excised, and were homogenized in 1 ml Trizol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). After incubation for 5 min, 0.2 ml chloroform was added and the mixture was incubated for 2 min. The mixture was centrifuged at 12,000 g for 15 min at 4˚C, and the upper phase was transferred to another tube. After addition of 0.5 ml isopropyl alcohol, the mixture was incubated for 10 min. Then the mixture was centrifuged at 12,000 g for 10 min at 4˚C. The supernatant was removed, and 1 ml of 75% ethanol was added to the RNA pellet. After centrifugation at 7500 g for 5 min at 4˚C, the supernatant was removed and the RNA pellet was air-dried. Then the RNA pellet was dissolved in nuclease-free water (Thermo Fisher Scientific, Waltham, MA, USA). The RNA concentration was determined by measuring the absorbance at 260 nm using a spectrophotometer, Smart Spec 3000 (Bio-Rad, Hercules, CA, USA).

TaqMan Gene Expression Assays for rat 11β-HSD1 (#Rn00567167_m1), rat 11β-HSD2 (#Rn00492539_m1), rat tumor necrosis factor (TNF) (#Rn999990- 17_m1), and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#Rn01- 7757-63_g1) were purchased from Applied Biosystems (Foster City, CA, USA). RT-PCR was performed using the Taq Man RNA-to-CT 1-Step Kit and Step One Plus Real Time PCR System (Applied Biosystems, Foster City, CA, USA). As a template, 20 ng RNA was used for each sample. RNA was mixed with 0.5 μl RT Enzyme Mix, 10 μl RT-PCR Mix, 1 μl Gene Expression Assay, and nuclease-free water (total volume 20 μl). The mixture was incubated for 15 min at 48˚C for reverse transcription, and for 10 min at 95˚C for activation of DNA polymerase. Then the mixture was incubated for 15 sec at 95˚C for denaturing and for 1 min at 60˚C for annealing/extension for 49 cycles. All reactions were carried out in triplicate. For normalization, the ratios of 11β-HSD1, 11β-HSD2, and TNF mRNA versus GAPDH mRNA were calculated in each sample.

For immunohistochemistry, 5 μl of 1 mg/ml LPS-PG was injected into the palatal gingiva around the second maxillary molars in three rats on day 1, day 3, and day 5, and rats were euthanized with an overdose of isoflurane and pentobarbital on day 9. After euthanasia, the rats were perfused with 10% formaldehyde neutral buffer solution (Sigma-Aldrich, St. Louis, MO, USA). The maxillary bones were excised and fixed in 10% formaldehyde neutral buffer solution at 4˚C for 7 days. Then the bones were decalcified in a rapid decalcification solution, K-CX (Falma, Tokyo, Japan) at 4˚C for 24 h, followed by conventional dehydration and paraffin embedding. After cutting into 5 μm-thick sections, the specimens were deparaffinized and stained with hematoxylin-eosin (HE) or immunostained with rabbit anti-11β-HSD1 monoclonal antibody (abcam, Cambridge, UK) and Envision + kit/HRP (DAB) (Dako, Glostrup, Denmark). Images were captured using an all-in one microscope, BZ-9000 (Keyence, Osaka, Japan).

One-way analysis of variance followed by a post-hoc test (Tukey) and significance testing of Pearson’s correlation coefficient were performed using SPSS software version 21.0 (IBM, Armonk, NY, USA).

In this study, experimental periodontitis was induced by intragingival injection of LPS from Porphyromonas gingivalis (LPS-PG) in rats. LPS-PG was injected into the palatal gingiva around the second maxillary molars on day 1, day 3, and day 5. Micro-CT analysis revealed alveolar bone loss around the second maxillary molar on day 14 (Figure 1(b)), demonstrating that experimental periodontitis was induced by LPS-PG injection.

The expression levels of 11β-HSD1, 11β-HSD2, and TNF mRNA in rat gingiva were analyzed using real-time RT-PCR methods as described above. As shown in Figure 2(a), the ratio of 11β-HSD1 mRNA versus GAPDH mRNA was significantly higher in gingiva injected with 1 mg/ml LPS-PG than in controls injected with endotoxin-free water. The ratio of 11β-HSD2 mRNA versus GAPDH mRNA was not significantly increased in LPS-PG-injected gingiva compared with controls (Figure 2(b)). The expression of TNF, a common inflammatory cytokine, was significantly higher in 1 mg/ml LPS-PG-injected gingiva than in controls (Figure 2(c)). As shown in Figure 3, the expression of 11β-HSD1 was positively and significantly correlated with that of TNF, in accordance with our previous report [21] .

The expression of 11β-HSD1 at the protein level was examined by immunostaining using anti-11β-HSD1 antibody. HE-staining showed that inflammatory cells such as neutrophils were infiltrated in LPS-PG-injected gingiva (Figure 4(b)). Immunostaining demonstrated that many round cells (presumably inflammatory cells such as neutrophils and monocytes) were positively stained with anti-11β-HSD1 antibody in LPS-PG-injected gingiva (Figure 4(d)).