Kazuo Todoki

Characterization by Electron Spin Resonance Spectroscopy of Reactive Oxygen Species Generated by Titanium Dioxide and Hydrogen Peroxide

The influence of reactive oxygen species (ROS) on the surface modification of titanium implants and osseointegration is unclear. The aim of this study was to evaluate the ability of titanium dioxide (TiO2) to generate ROS in the presence of H2O2 and to determine whether any ROS thus generated play a role in osseointegration, as measured by electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrolline-N-oxide (DMPO). We demonstrate that TiO2 together with H2O2 generated hydroxyl radicals (HO•), as shown by a time-dependent increase in the spin concentration of the ESR signal for the DMPO-OH spin adduct, indicating HO• generation. Interestingly, irradiated TiO2 with H2O2 generated the superoxide (O2 •-), as shown by an increase in the spin concentration of the signal for the DMPO-OOH spin adduct, indicating O2 •- generation during the period of irradiation (0–5 min). These results suggest that ROS generated from the TiO2 layer may be involved in creating appropriate conditions for the osseointegration of dental implants into alveolar bone tissues.

Direct Assessments of the Antioxidant Effects of Propofol Medium Chain Triglyceride/Long Chain Triglyceride on the Brain of Stroke-prone Spontaneously Hypertensive Rats Using Electron Spin Resonance Spectroscopy

Background:Antioxidant anesthetics such as propofol (2,6-diisopropylphenol) directly inhibit lipid peroxidation via the generation of reactive oxygen species. Currently, there are no other studies regarding the direct effects of propofol medium chain triglyceride/long chain triglyceride (MCT/LCT) on reactive oxygen species generation or in experimental models of reactive oxygen species–induced oxidative stress in the brain. Methods:The authors investigated the effects of propofol MCT/LCT on reactive oxygen species (hydroxyl radical or superoxide) by electron spin resonance spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide. The effects of propofol MCT/LCT on oxidative stress in the brain of Wistar-Kyoto rats or stroke-prone spontaneously hypertensive rats were investigated by using an in vivo L-band electron spin resonance system to monitor the decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl as a nitroxyl spin probe. Results:These studies provided direct evidence that propofol MCT/LCT inhibited hydroxyl radical generation, but not superoxide generation. Regarding the hydroxyl radical from the Fenton system, it is likely to be due to the scavenging effects of vehicle. Anesthesia with propofol MCT/LCT reduced the degree of the high oxidative stress in the brain of stroke-prone spontaneously hypertensive rats. Conclusion:The current data show that propofol, mixed with clinical reagents (propofol MCT/LCT), resulted in the down-regulation of high oxidative stress due to scavenging hydroxyl radical, as demonstrated by in vitro or in vivo electron spin resonance analysis. These results led to reduced levels of hydroxyl radical, formed by brain injury such as stroke, and may therefore provide advantages for neuroprotection during anesthesia for craniotomy, e.g., in cases of brain disease.

Direct pharmacological action of vasoactive substances on pulpal blood flow: An analysis and critique*†

An adequate blood supply to the dental pulp is essential to the health of the tooth; therefore, there have been a number of efforts to study pulpal blood flow and the factors which influence it. However, blood flow to the dental pulp is relatively inaccessible and apparently quite low. Consequently, it is difficult to obtain accurate flow measurements, partly owing to methodological difficulties with the small size of the tissue and its enclosure within rigid walls. In this study, the effects of locally applied vasoactive substances and their specific antagonists on pulpal blood flow have been examined by laser Doppler flowmetry. It is the purpose of this article to examine, in-depth, the involvement of endogenous vasoactive substances in the regulatory mechanism of blood flow within the dental pulp and expand our knowledge of pulpal microcirculatory hemodynamics.

Gingival vascular functions are altered in type 2 diabetes mellitus model and/or periodontitis model

The association of vascular reactivity between diabetes and periodontal 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 + Porphyromonas 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-tetramethylpyrrolidin-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 hyperemia 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 diabetes mellitus and/or periodontal disease animal models due to increasing oxidative stress in the gingival circulation.

Nitric oxide levels in rat hypothalamus are increased by restraint stress and decreased by biting

Abstract Mastication, which includes biting, is of great importance not only for the intake of food but also for the mental, physical and physiological functioning of the body. For example, biting suppresses the stress response. Although biting and nitric oxide (NO) appear to modulate brain dynamics during stress, the underlying mechanisms have not been elucidated. In this study, we examined the effect of biting during restraint stress on NO levels in the rat hypothalamus. To this end, we used NO-selective electrodes that were calibrated by electron spin resonance (ESR) spectroscopy. We implanted the electrodes and probes for perfusion of solutions into the brain of rats, near the hypothalamus. Saline containing 10 mM N-nitro-L-arginine methyl ester (L-NAME), which is one of the most commonly used inhibitors of nitric oxide synthase (NOS), was employed as the perfusate. L-NAME prevented increases in NO levels in the rat hypothalamus that were induced by restraint stress and biting. Hypothalamic NO levels in rats under restraint stress for 180 min were increased above levels observed in unrestrained control rats. The increase in hypothalamic NO (from 2.123 μM to 4.760 μM) during restraint stress was reduced after biting for 30 min. The decay rate of NO levels after biting was −0.584 pA/min (−0.071 μM/min). We conclude that: (i) it is possible to evaluate NO levels in vivo in rat brain; (ii) NO levels are increased by restraint stress; and (iii) this increase is prevented by biting behavior.

Role of nitric oxide in post-ischemic gingival hyperemia in anesthetized dogs

Abstract The possible involvement of nitric oxide (•NO) in the preservation of blood flow to the canine gingiva after compression of gingival tissue was studied. Gingival blood flow, gingival tissue oxygen partial pressure (PO2), external carotid arterial blood pressure and external carotid arterial blood flow were monitored before, during, and after compression of gingival tissue in the presence and absence of the nitric oxide synthase inhibitor, Nω-nitro-L-arginine-methyl-ester (L-NAME). Compression of gingival tissue resulted in an immediate decrease in gingival blood flow and tissue PO2. After the compression of gingival tissue, hyperemia was observed in the gingiva, which depended on the duration of ischemia. Gingival tissue PO2 slowly recovered during hyperemia. Pretreatment with L-NAME (60 mg/kg, i.a.) significantly suppressed reactive hyperemia in gingival tissue. The L-NAME-suppressed reactive hyperemia was partially reversed by treatment with L-arginine (60 mg/kg, i.a.). In addition, •NO was detected using an •NO selective electrode during interruption of blood flow and during reactive hyperemia in the gingiva. These results suggest that •NO contributes to the vasodilation during reactive hyperemia in gingival tissue, and aids in the maintenance of homeostasis in gingival circulation.

Inhibitory effects of Jixueteng on P. gingivalis-induced bone loss and osteoclast differentiation

OBJECTIVE The aim of this study was to investigate the possibility of Jixueteng as a preventive and therapeutic drug for the periodontitis. We investigated the inhibitory effects of Jixueteng on Porphyromonas gingivalis-induced bone loss in mice, antibacterial activity against P. gingivalis and differentiation of osteoclast and viability of cells. MATERIALS AND METHODS Fifty-four male, 4-week-old C57BL/6N mice, were randomly divided into the following three groups of 18 mice each; group A served as the P. gingivalis non-infected control (sham group), group B was infected orally with P. gingivalis and group C was administered Jixueteng extract in drinking water and was then infected with P. gingivalis. In order to evaluate the effect of Jixueteng, the distance from the alveolar bone crest to the cemento-enamel junction was determined. P. gingivalis suspension was exposed for 1, 15 and 60 min to 5 ml of the Jixueteng extract. Furthermore, to clarify the mechanism of the inhibitory effects of Jixueteng on osteoclast formation, Jixueteng extract was added to the culture of mouse bone marrow cells, osteoclast precursor. RESULTS Administration of Jixueteng along with P. gingivalis infection significantly reduced alveolar bone loss compared to P. gingivalis infection. Jixueteng treatment at the concentration of 0.01% significantly inhibited osteoclast formation. The addition of Jixueteng extract (0.1%, 0.01%, and 0.001%) to the culture showed a significant inhibition of the number of surviving osteoclasts in a dose-dependent manner. CONCLUSION Jixueteng has an antibacterial activity against P. gingivalis and inhibitory effects on osteoclastogenesis, it may be useful as a therapeutic drug in the treatment of P. gingivalis-induced periodontitis.

A biological study establishing the endotoxin limit of biomaterials for bone regeneration in cranial and femoral implantation of rats.

The purpose of this study was to accurately quantify the risk of endotoxin contamination in biomaterials for bone regeneration in order to establish the acceptable endotoxin limit. Collagen sheets containing varying amounts of purified endotoxin from Escherichia coli and dried, heat-killed E. coli or Staphylococcus aureus cells were implanted into cranial or femoral defects in rats. These defects were artificially prepared to a size of 5 × 5 mm or a diameter of 1 mm, respectively. The degree of osteoanagenesis was assessed by soft X-ray radiography and histopathology at 1 and 4 weeks after implantation. The collagen sheet containing the dried E. coli cells showed a dose-dependent delay in cranial and/or femoral osteoanagenesis at endotoxin activities of more than 33.6 EU/mg, at which no inflammatory response was observed. In contrast, no such observation occurred with the collagen sheet containing S. aureus cells. These results suggest that endotoxins may affect the process of osteoanagenesis. Additionally, the no-observed-adverse-effect level was 9.6 EU/mg, corresponding to 255 EU/kg body weight in rats. Interestingly, no delay in osteoanagenesis was induced by the implantation of collagen sheets containing purified endotoxin at any dose tested. This suggested that pure endotoxin implanted into tissues having poor circulation of bodily fluids without bleeding may not be recognized as a foreign substance and may not induce a significant biological response.

Contribution of nitrergic nerve in canine gingival reactive hyperemia

Reactive hyperemia reflects a compensatory vasodilation response of the local vasculature in ischemic tissue. The purpose of this study is to clarify the mechanism of regulation of this response in gingival circulation by using pharmacological analysis of reactive hyperemia and histochemical analysis of gingival tissue. Application of pressure to the gingiva was used to create temporary ischemia, and gingival blood flow was measured after pressure release. Reactive hyperemia increased in proportion to the duration of pressure. Systemic hemodynamics remained unaffected by the stimulus; therefore, the gingival reactive hyperemia reflected a local adjustment in circulation. Gingival reactive hyperemia was significantly suppressed by nitric oxide (NO) synthase inhibitors, especially the neural NO synthase-selective antagonist 7-nitroindazole, but not by anticholinergic drugs, β-blockers, or antihistaminergic drugs. Moreover, immunohistochemical staining for neural NO synthase and histochemical staining for NADPH diaphorase activity were both positive in the gingival perivascular region. These histochemical and pharmacological analyses show that reactive hyperemia following pressure release is mediated by NO-induced vasodilation. Furthermore, histochemical analysis strongly suggests that NO originates from nitrergic nerves. Therefore, NO may play an important role in the neural regulation of local circulation in gingival tissue ischemia.

Microcirculation: function and regulation in microvasculature

The primary purpose of microcirculation is to transport nutrients and oxygen and to remove metabolic waste products from the tissue. It is also well known that the fundamental mechanism for vascular control is the local regulation of the basal vascular tone, which is reinforced by blood pressure and counteracted by tissue metabolites. Thus, the wellbeing of the tissue depends on the circulatory transport process, which is governed by many functional parameters of the microcirculation, such as blood flow, blood volume, intravascular and extravascular pressures, and capillary permeability (Aukland and Nicolaysen, 1981; Rothe and Friedman, 1976; Kety, 1960); and therefore the microvasculature is the site of control of tissue perfusion, blood-tissue exchanges and tissue blood volume. Each of these functions can be associated with specific microvascular segments. Because intravascular pressure measurements demonstrate that a major fraction of total pressure dissipation occurs in precapillary microvessels, the arterioles are designated as resistance vessels. At any given moment local and extrinsic stimuli impinge on the wall of the arteriole and exert control over the calibre of the microvessel. In so doing these signals modulate the blood flow through the tissue. The capillaries are the major exchange vessels; across the surface of the microvessels flow all the nutrients required to sustain the cells of the body. In most tissues only a fraction of the capillaries (precapillary sphincter) are perfused under normal conditions and exert control over the number of perfused capillaries. Finally, the venules are classified as capacitance vessels because most of the tissue blood volume is localized in these microvessels.