Fig. 3. Alterations in gingival reactive hyperemia in Ws, Ws + Pg, GKs and GKs + Pg groups from 7 to 31 weeks old. Peak (A), T1/2 (B) and Mass (C) in gingival reactive hyperemia were plotted against time. Inserted figures represent regression lines fitted to a typical plot of laser Doppler flowmeter data obtained from Ws, Ws + Pg, GKs and GKs + Pg. The columns show slope of parameters against time. Data are presented as mean ± SEM. (n = 5 in each group). *p<0.05 vs Ws. †p<0.05 vs GKs. #p<0.05 vs Ws + Pg. is associated with plasma TNF-α levels in subjects with type 2 diabetes support the hypothesis that periodontal infection and inflammation may contribute to insulin resistance.(21) Elevated TNF-α was observed in non-obese diabetes patients.(22) Numerous studies have long recognized that periodontitis is more prevalent in diabetic patients and worsens with the development of diabetes;(23) periodontal disease has been called as the sixth complication of diabetes.(24) A previous epidemiological survey showed that incident tooth loss caused by periodontal disease was significantly associated with peripheral arterial disease.(25) This indicates that vascular reactivity consisting of endothelial and smooth muscle functions is suppressed in the presence of circula- tory failure such as in periodontal disease and diabetes. Oral S. Sugiyama et al. challenge with Pg in Ws and GKs enhanced alveolar bone resorp- tion, with the effect significantly enhanced in GKs (Fig. 1). In vivo L-band ESR spectroscopy with an ESR imaging system is a highly useful technique for the non-invasive investigation of oxidative stress induced by ROS in living organisms. Several previous studies have demonstrated that quantitative ESR analysis using the spin probe C-PROXYL can be used to investigate redox status under conditions of oxidative stress in the skin of the maxillofacial region in mice.(10,11,18,20) The present study utilized in vivo L-band ESR to evaluate oxidative stress induced by Pg infection or diabetes in the oral and maxillofacial region of the rat. These results firstly showed that the ESR technique employed herein could be a powerful tool to selectively detect free radicals, including oxidative stress, and to monitor free radical reactions in vivo in the oral and maxillofacial region in the rat model (Fig. 2). We showed that the decay rate constant (K1) of C-PROXYL was significantly greater in the oral and maxillofacial region of GKs + Pg compared to Ws, Ws + Pg, and GKs (Fig. 2D). In a previous study using L-band ESR analysis, we reported that the metabolism of lipophilic nitroxyl spin probes in the brain can be divided into phase I (vascular system) and phase II (reduced excretion via the kidney), according to a two-compartment model of distribution.(5,10,11) The present study indicated that the metabo- lism of C-PROXYL in the oral and maxillofacial region of the rat also occurred according to a two-compartment model (K1 and K2; Fig. 2 A–C). The decay of the nitroxyl spin probe, C-PROXYL, which is reflected in ESR signal intensity in the oral and maxillo- facial region, also appears to follow the usual pharmacokinetic pattern for excretion of drugs: those that tend to stay in the vascular system are excreted very rapidly (phase I; K1), while C- PROXYL show much slower excretion via the kidney (phase II; K2).(5,11,18) When Ws were challenged with Pg the level of oxida- tive stress in the oral and maxillofacial region was not signifi- cantly increased in comparison to that in non-treated animals. Taken together, it would be likely that the level of oxidative stress would be aggravated by Pg infection of the oral and maxillofacial region of GKs, suggesting that periodontitis may increase oxida- tive stress in the microcirculation in the oral and maxillofacial region of the diabetes model. Diabetes mellitus, a major risk factor for vascular diseases, is associated with accelerated atherosclerosis and a high rate of arterial thrombotic complications such as retinopathy, nephropathy and cerebrovascular disease.(1) In diabetes, angiopathies can occur in both capillaries and large vessels. Periodontal disease is a well-known complication of diabetes, and persons with diabetes are at increased risk of developing severe periodontitis.(26–28) In addition, it has been reported that diabetes-induced insulin resis- tance causes endothelial dysfunction in blood vessels.(3,4) Reactive hyperemia in the gingival circulation was significantly increased in Ws after treatment with ACh and NTG. However, those effects were not observed in GKs after treatment with ACh and NTG (Fig. 4). These results suggest that the gingival microvascular function could reflect the state of the microcirculation in other vascular beds, including those in persons with diabetes mellitus. Endothelium-dependent vasodilation is abnormal in forearm resistance vessels of patients with insulin-dependent diabetes mellitus. This abnormality may be relevant to the high prevalence of vascular disease that occurs in persons with insulin-dependent diabetes mellitus.(29) In our study, we found that gingival vascular reactivity was reduced in animals challenged with Pg and that this reduction was enhanced in GKs (Fig. 3). Application of ACh and NTG to gingival mucosa did not affect GBF in 10-week-old GKs (Fig. 4). Therefore, the alteration in gingival vascular endo- thelial and smooth muscle functions would occur after 10 weeks in GKs and GKs + Pg. In conclusion, results of the present study indicate that peri- odontal disease reduced gingival vascular reactivity and decreased reactive hyperemia, and this effect was accelerated by diabetes J. Clin. Biochem. Nutr. | September 2012 | vol. 51 | no. 2 | 111 ©2012 JCBN