Fig. 2. Assessment of oxidative stress in the oral and maxillofacial region on 10-week-old Ws, Ws + Pg, GKs and GKs + Pg. All rats were anesthetized with pentobarbital (45 mg/kg, i.p.). L-band ESR was used to determine the signal decay of C-PROXYL in the oral and maxillofacial region of all rats. Decay rate constants of (K1 and K2) C-PROXYL in the oral and maxillofacial region of 10-week-old Ws (circles in A), Ws + Pg (circles in B), GKs (circles in C) and GKs + Pg (triangles in A–C). The columns represent: K1 of C-PROXYL in the oral and maxillofacial region of 10-week-old Ws, Ws + Pg, GKs and GKs + Pg (D). Results are expressed as mean ± SD in all groups (n = 4 in each group). *p<0.05 vs Ws. †p<0.05 vs GKs. #p<0.05 vs Ws + Pg. mg/dL; GKs + Pg, 283.4 ± 14.4 mg/dL to 365.0 ± 16.5 mg/dL). Effects of alveolar bone resorption on periodontal tissue was enhanced in Ws and GKs orally challenged with Pg, with this effect being significantly enhanced by Pg infection in GKs (Fig. 1). Evaluation of oxidative stress in the oral and maxillofacial region in diabetes and/or periodontal disease models. The 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (C-PROXYL) is a suitable spin probe for the study of free radical reactions in the rodent skin by in vivo ESR detection.(11,18,20) These methods were also applied for the evaluation of oxidative stress of the oral and maxillofacial region in Ws, Ws + Pg, GKs and GKs + Pg. In the experiments with the oral and maxillofacial region of Ws, Ws + Pg, GKs and GKs + Pg after i.v. treatment with C- PROXYL, we initially assessed the distribution of C-PROXYL using the L-band ESR technique. With this method, we succeeded in measuring oxidative stress as decay rate constant (K1 and K2) of C-PROXYL in the oral and maxillofacial region of the animal models (Fig. 2 A–C). The decay rate constant (K1) of C-PROXYL was significantly greater in the oral and maxillofacial region of GKs + Pg compared to the Ws, Ws + Pg and GKs groups (Fig. 2D). These results suggested that increased oxidative stress occurs in the oral and maxillofacial region of the periodontal disease and/or diabetes rat models. Evaluation of vascular responses using gingival reactive hyperemia in the diabetes and/or periodontal disease model. In experiments using non-infected Ws, compression of gingival tissue for 1 min resulted in an immediate decrease in GBF. One minute after release of gingival tissue compression, reactive hyperemia was observed in the gingiva. Peak, T1/2, and Mass of GBF were increased by gingival compression for 1 min. 110 This effect was significantly enhanced by topical oral mucosal administration of ACh and NTG. Cardiac function was not influenced by gingival reactive hyperemia or pharmacological reagents (data not shown). These results suggest that gingival reactive hyperemia could be measured as a vascular endothelial response that did not have an effect on the systemic circulation. In Ws that were orally challenged with Pg there was a remark- able reduction in gingival reactive hyperemia throughout the experimental period, which was approximately 6 months. The reduction of each parameter analyzed in Pg-challenged GKs was significant, while only the reduction in Peak was significant in Ws. Pg-challenged rats, although reductions were noted in the other two parameters analyzed (Fig. 3). As shown in Fig. 4, Peak, T1/2 and Mass were all significantly increased in Ws and Ws + Pg compared to GKs and GKs + Pg after administration of ACh (A) or NTG (B) on palatal mucosa, suggesting that the effect of ACh and NTG was reduced by Pg infection of Ws. Application of ACh and NTG on the palatal mucosa did not affect reactive hyperemia in the GKs and GKs + Pg groups, indicating that the gingival circulation as shown by the gingival vascular response to ACh and NTG was reduced in GKs and GKs + Pg compared to Ws and Ws + Pg. Discussion Periodontitis is a common, chronic inflammatory disease initiated by bacteria and has increased prevalence and severity in patients with type 2 diabetes.(1) Current evidence indicated that there is a bidirectional interrelationship between diabetes and inflammatory periodontitis. The findings that chronic periodontitis doi: 10.3164/jcbn.11-103 ©2012 JCBN