Animals were housed in groups of 5 per cage in a room maintained under standardized light (12:12 h light-dark cycle) and temperature (22 ± 3°C) conditions with free access to food pellets and tap water. The animals were divided into 4 groups: Group 1, Porphyromonas gingivalis (Pg)-non-infected Ws; Group 2, Ws orally challenged with Pg (Ws + Pg); Group 3, Pg-non-infected GKs, and Group 4, GKs orally challenged with Pg (GKs + Pg). Each group was comprised of 5 rats. Bacteria and culture conditions. The bacterial strain used was Pg American Type Culture Collection (ATCC) 33277. Pg strains were grown at 37°C for 18 h in an anaerobic chamber (Anaerobox, Hirasawa, Tokyo, Japan) with an atmosphere of 85% N2, 10% H2, and 5% CO2 in brain heart infusion broth (Difco, Detroit, MI) supplemented with 5 mg/mL yeast extract, 5 μg/mL hemin, and 0.2 μg/mL vitamin K1. Porphyromonas gingivalis infection and alveolar bone loss in rats. Rats were given sulfamethoxazole (1 mg/mL) and trimethoprim (200 μg/mL) in drinking water for 7 days ad libitum to reduce the original oral flora, followed by a 3-day antibiotic-free period before Pg infection. Each rat received 0.5 mL (1.5 × 109 cells per mL) Pg ATCC 33277 suspended in 5% carboxymethyl- cellulose (Sigma Chemical, St. Louis, MO) by oral gavage 3 times/week at the ages of 5, 6, 17, and 32 weeks. One aspect of periodontal disease is the resorption of alveolar bone. After sacrifice at the age of 33 weeks, the upper jaws were defleshed after 10 min in an autoclave at 15 pounds/inch2 and immersed in 3% hydrogen peroxide, rinsed, air dried, and stained with 1% methylene blue. Horizontal bone loss around the maxillary molars was assessed morphometrically. The distance from the cement- enamel junction (Fig. 1, white line) to the alveolar bone crest (Fig. 1, arrowhead) was measured at 7 buccal sites per rat. Measurements were made under a dissecting microscope (×40) fitted with a digital high-definition system (Digital HD micro- scope VH-7000, KEYENCE, Osaka, Japan) standardized to give measurements in millimeters.(17) In vivo ESR measurement. Ws and GKs were anesthetized with pentobarbital (45 mg/kg, i.p.) at the age of 11 weeks. We then analyzed the oral and maxillofacial region of Ws or GKs with in vivo L-band ESR analysis as described previously.(5,6–11,18) The ESR spectra from Ws and GKs were obtained with an L-band ESR spectrometer (JES-RE-3L; JEOL, Akishima, Japan) equipped with a 4 window loop-gap resonator under the following condi- tions: microwave power, 10 mW; magnetic field, 50.0 ± 10 mT; field modulation width, 0.1 mT; receiver gain, 79–320; sweep time, 0.5 min; and time constant, 0.03 s. All experiments were repeated a minimum of 4 times. Gingival blood flow. Animals were anesthetized with sodium pentobarbital (45 mg/kg, i.p.) and were subsequently given small maintenance doses as necessary. After body weights were determined, A tail vein was tapped with 25 gauge needle, and a glucose levels was determined by a FreeStyle Freedom Glucose Meter (Nipro, Osaka, Japan). Gingival blood flow (GBF) was measured at the palatal gingiva by a laser Doppler flowmeter (TBF-LN1, Unique Medical Co. Ltd., Tokyo, Japan) with a laser Doppler probe (diameter 2.0 mm). Heart rate was monitored to determine the effect on systemic hemodynamics by agents administered or gingival reactive hyperemia. To evaluate vascular endothelial and smooth muscle functions, 100 mg/mL acethyl- choline (ACh) or 5 mg/mL nitroglycerine (NTG) was topically absorbed from an area of gingival mucosa approximately 2 mm in diameter for 1 min before gingival compression for 1 min. Reactive hyperemia was elicited at the same place as ACh and NTG application by release of occlusive gingival compression by the laser Doppler probe for 1 min. The reactive hyperemia in the gingival circulation was estimated by basal blood flow (Basal), maximum response (Peak), time taken for the maximum response to fall to one half (T1/2), and increased total amount of blood flow (Mass).(19) The output signals from the flowmeter were recorded S. Sugiyama et al. on a computer hard disc through an A/D converter and displayed simultaneously on the monitor. Recorded GBF was analyzed using data analysis software (Chart ver. 4.2 AD Instruments, Inc., Colorado Springs, CO). These GBF measurements were performed twice a month on the rats when they were from 7 to 31 weeks old. The animals were returned to the same cage after GBF measure- ments. Statistical analysis. Analysis of variance and multiple com- parison tests using the Tukey’s method were applied to determine differences among the 4 groups. When comparing only one pair, we used the Student’s unpaired t test. Data are expressed as the mean ± SEM. Results Effects of alveolar bone resorption on periodontal tissue. Plasma glucose levels of Ws and GKs were determined once every 2 weeks from 7 to 31 weeks after birth. The plasma glucose levels were significantly decreased during the experimental period in the Ws groups (Ws, 113.6 ± 6.8 mg/dL to 101.3 ± 3.5 mg/dL; Ws + Pg, 113.6 ± 4.6 mg/dL to 103.0 ± 7.5 mg/dL), while the plasma glucose levels in the GKs groups were elevated during the experimental period (GKs, 281.6 ± 14.7 mg/dL to 315.3 ± 14.8 Fig. 1. Measurement of bone loss in an upper jaw from 4 animals in each group. Morphometric bone levels in 33-week-old rats (A). The distance from the cement enamel junction (arrowhead in A) to the alveolar bone crest (white line in A) was measured at 7 buccal sites per rat. (B) Data are presented as mean ± SEM. (n = 4 in each group). *p<0.05 vs Ws. †p<0.05 vs GKs. #p<0.05 vs Ws + Pg. J. Clin. Biochem. Nutr. | September 2012 | vol. 51 | no. 2 | 109 ©2012 JCBN