Original Article GjtJ01KOchoC980ibyreu18.iBno3gnrS20n1tNi-ona1605,cal-4g0iJ1lo/e08aj0Aftcp96y3biCratfnivloci.n1rl ei1Fac-ra1el0elB3Rioavcdhiceaaml iRsstercsyeaunrcdhNlJauaptrairtnionfunctions are altered in type 2 diabetes mellitus model and/or periodontitis model Shuta Sugiyama,1,† Shun-suke Takahashi,1,† Fumi-aki Tokutomi,1 Ayaka Yoshida,1 Kyo Kobayashi,1 Fumihiko Yoshino,1 Satoko Wada-Takahashi,1 Toshizo Toyama,2 Kiyoko Watanabe,2 Nobushiro Hamada,2 Kazuo Todoki3 and Masaichi-Chang-il Lee1,* 1Department of Clinical Care Medicine, Division of Pharmacology and ESR Laboratories, Kanagawa Dental College, 2Department of Infection Control, Division of Microbiology, Kanagawa Dental College and 3Department of Dental Hygiene, Division of Oral Science & Pharmacology, Shonan Junior College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan (Re9ceived 10 August, 2011; Accepted 22 November, 2011; Published online 30 March, 2012) svCTthirde2TCeieca0hoadtt1eipssvdt2syhoeireuscigisoaCaehnrto,itigomod©inpinmse2aotnorl0finw1bavs2ucaoctsrJiAeckoCusntsiBlta,sraiNrapbrrntrueiodctaiplocertneridpvlyiirLstotycirdciibtuebeecnudttst.iweod,eneuwinnhddiaecianrhbythepmeteetersemdariminutdsmspo,uefpnrtrihooee-- dontal disease has not been clarified. Gingival blood flow was measured by laser Doppler flowmetry for 31 weeks in Wistar rats, Wistar rats orally challenged with Porphyromonas gingivalis (Wistar rats + Porphyromonas gingivalis), Goto-Kakizaki rats, and Goto-Kakizaki rats orally challenged with Porphyromonas gingivalis (Goto-Kakizaki rats + Porphyromonas gingivalis). Effects of alveolar bone resorption on periodontal tissue was enhanced in Wistar rats + Porphyromonas gingivalis, and Goto-Kakizaki rats, with this effect being significantly enhanced by Goto-Kakizaki rats + Por- phyromonas gingivalis. Using the L-band electron spin resonance technique, we succeeded in measuring oxidative stress as decay rate constant (K1 and K2) of 3-carbamoyl-2,2,5,5-tetramethylpyrro- lidin-1-yloxy in the oral and maxillofacial region of the animal models. The decay rate constant (K1) of 3-carbamoyl-2,2,5,5- tetramethylpyrrolidin-1-yloxy was significantly greater in the oral and maxillofacial region of Goto-Kakizaki rats + Porphyromonas gingivalis compared to Wistar rats, Wistar rats + Porphyromonas gingivalis and Goto-Kakizaki rats groups. Gingival reactive hyper- emia was attenuated by periodontal disease, and this effect was also remarkable in the diabetes mellitus model. Taken together, we found that vascular endothelial function was decreased in dia- betes mellitus and/or periodontal disease animal models due to increasing oxidative stress in the gingival circulation. Key Words: gingival circulation, oxidative stress, L-band ESR, diabetes mellitus, periodontitis T species (ROS) on inflammatory pathways in the pathogenesis TIhnetirmodpourctatniocne of oxidative stress induced by reactive oxygen of type 2 diabetes and periodontitis has recently received atten- tion.(1) Oxidative stress occurs when the production of ROS exceeds the capacity of the cell to detoxify these potentially inju- rious oxidants using endogenous antioxidant defense systems. Conditions associated with oxidative stress include ischemia/ reperfusion, hypercholesterolemia, diabetes, and hypertension. The adhesion of circulating blood cells (leukocytes, platelets) to vascular endothelium is a key element in the pro-inflammatory and prothrombogenic phenotype assumed by the vasculature in these and other disease states that are associated with oxidative stress. There is a growing body of evidence that links the blood cell-endothelial cell interactions in these conditions to the enhanced production of ROS.(2) That an increase in insulin resistance due to diabetes mellitus causes a reduction of the nitric oxide (NO) production by endothelium dysfunction and arterial sclerosis has been demonstrated.(3,4) In research on the correlation of diabetes doi: 10.3164/jcbn.11-103 ©2012 JCBN with periodontitis, epidemiologic studies are largely performed, but little has been reported using a basic approach to clarifying the relationship between these conditions, especially from the aspects of oxidative stress and vascular reactions. Therefore, the mecha- nism of why diabetes aggravates periodontal disease is still unclear. We previously developed an electron spin resonance (ESR)- based technique for the detection of free radical reactions, including ROS, in biological systems.(5–11) Nitroxyl radicals are very useful as exogenous spin probes for measuring free radical distribution, oxygen concentration, and redox metabolism by in vivo ESR in biological systems.(5,9–12) It has been reported that nitroxyl radicals, referred to as ‘nitroxyl spin probes’, lose their paramagnetism through a redox reaction when exposed to a reducing agent in biological systems.(13,14) The signal decay rate of the nitroxyl spin probe provides evidence of ROS generation and changes in redox status in biological systems.(15,16) However, no other study has specifically measured the signal decay rate as oxidative stress in experimental models of ROS-induced oxidative stress in the oral region. This study was performed based on periodontal vascular events in a diabetic animal model, during which oxidative stress on the oral circulation occurred. We determined that gingival reactive hyperemia reflected vascular endothelial function through analysis of reactive hyperemia of the gingival tissue in animal models of diabetes mellitus and/or periodontitis with vascular disease. We succeeded through the use of an ESR in vivo technique and laser Doppler flowmetry (LDF) in evaluating gingival reactive hyper- emia and found that vascular endothelial function was decreased in diabetes mellitus and/or periodontal disease animal models due to increasing oxidative stress in the gingival circulation. Materials and Methods Animals. In this study, Wistar rats (Ws) were used as control animals and Goto-Kakizaki rats (GKs) were used as a type 2 diabetes mellitus model. For both groups, 4-week-old male animals were purchased from CLEA Japan, Inc. (Tokyo, Japan). The procedures used in this study were in accordance with the guidelines of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication NO. 85–23, revised 1985). Also, Institutional Animal Care Committees approved our protocols. *To whom correspondence should be addressed. E-mail: ieeman@kdcnet.ac.jp †These authors contributed equally to this work. J. Clin. Biochem. Nutr. | September 2012 | vol. 51 | no. 2 | 108–113