Porphyromonas gingivalis infection modifies oral microcirculation and aortic vascular function in the stroke-prone spontaneously hypertensive rat (SHRSP).

The functional modulation of vascular endothelial cells associated with stroke and periodontal disease has not yet been clarified. The objective of this study is to analyze the vascular endothelial function of periodontitis and stroke animal models. We examined endothelial function and gingival blood flow in oral microcirculation in vivo and measured the isometric tension in vitro of the aorta in animal models for lifestyle-related diseases, such as periodontitis and stroke. Gingival reactive hyperemia (GRH) was measured using laser Doppler flowmetry. Wistar Kyoto rats (WKY) were used as control animals; Porphyromonas gingivalis (P. gingivalis) infected WKY (WKY + Pg) as the periodontitis model; stroke-prone spontaneously hypertensive rat (SHRSP) as the stroke model; and a final group consisting of P. gingivalis infected SHRSP (SHRSP + Pg). Furthermore, for each group, the relaxation of descending aortic ring preparations was measured using a force transducer. The GRH was estimated by maximum response (peak), time taken for the maximum response to fall to one half (T1/2), and increased total amount of blood flow (mass). The relative change in T1/2 and mass increased in SHRSP + Pg compared to WKY. However, mass significantly increased in WKY (758.59 ± 88.21 ml/min/100 g s to 1755.55 ± 226.10 ml/min/100 g s) and SHRSP (1214.87 ± 141.61 ml/min/100 g s to 2674.32 ± 675.48 ml/min/100 g s) after treatment with acetylcholine. In addition, T1/2 and mass significantly increased in WKY + Pg (624.18 ± 96.36 ml/min/100 g s to 2629.90 ± 612.01 ml/min/100 g s) and SHRSP + Pg (1116.36 ± 206.24 ml/min/100 g s to 1952.76 ± 217.39 ml/min/100 g s) after treatment with nitroglycerin. Furthermore, the endothelium-dependent relaxation of ring preparations, evoked by acetylcholine, was attenuated in SHRSP compared with WKY, but not in SHRSP + Pg. This attenuation effect in SHRSP could be prevented by superoxide dismutase pretreatment. Our results suggest altered endothelial function may occur in gingival tissue in animal models experiencing both periodontitis and stroke. Therefore, these results indicate the disruption of vascular function in oral microcirculation may be caused by the interaction between the oxidative stress induced by periodontitis and nitric oxide in periodontitis, similar to the interactions present in stroke cases.

Protective effects of (6R)-5,6,7,8-tetrahydro-l-biopterin on local ischemia/reperfusion-induced suppression of reactive hyperemia in rat gingiva.

We herein investigated the regulatory mechanism in the circulation responsible for rat gingival reactive hyperemia (RH) associated with ischemia/reperfusion (I/R). RH was analyzed using a laser Doppler flowmeter. RH and I/R were elicited by gingival compression and release with a laser Doppler probe. RH increased in a time-dependent manner when the duration of compression was between 30 s and 20 min. This increase was significantly suppressed by N (ω)-nitro-l-arginine-methyl-ester (l-NAME), 7-nitroindazole (7-NI), and 2,4-diamino-6-hydroxypyrimidine (DAHP). However, RH was markedly inhibited following 60 min of compression. This inhibition was significantly decreased by treatments with superoxide dismutase (SOD), (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4), and sepiapterin. The luminescent intensity of superoxide anion (O2 (•-))-induced 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a] pyrazine-3-one (MCLA) was markedly decreased by SOD and BH4, but only slightly by sepiapterin. BH4 significantly decreased O2 (•-) scavenging activity in a time-dependent manner. These results suggested that nitric oxide (NO) secreted by the nitrergic nerve played a role in regulating local circulation in rat gingiva. This NO-related regulation of local circulation was temporarily inhibited in the gingiva by the I/R treatment. The decrease observed in the production of NO, which was caused by suppression of NO synthase (NOS) activity subsequent to depletion of the NOS co-factor BH4 by O2 (•-), played a partial role in this inhibition.

Low-Intensity Pulsed Ultrasound Prevents Development of Bisphosphonate-Related Osteonecrosis of the Jaw-Like Pathophysiology in a Rat Model.

We developed a rat model of bisphosphonate-related osteonecrosis of the jaw (BRONJ) by removing a maxillary molar tooth (M1) from ovariectomized rats after treatment with alendronate. To mimic periodontitis, some of the rats were administered Porphyromonas gingivalis (p. gingivalis) at the M1 site every 2 to 3 d for 2 wk. Rats pretreated with alendronate plus p. gingivalis showed delayed healing of socket epithelia, periosteal reaction of alveolar bone formation and lower bone mineral density in the alveolus above adjacent M2 teeth. These abnormalities were prevented by tooth socket exposure to 20 min/d low-intensity pulsed ultrasound (LIPUS), which restored diminished expression of RANKL, Bcl-2, IL-6, Hsp70, NF-κB and TNF-α messenger ribonucleic acids in remote bone marrow, suggesting LIPUS prevented development of BRONJ-like pathophysiology in rat by inducing systemic responses for regeneration, in addition to accelerating local healing. Non-invasive treatment by LIPUS, as well as low-level laser therapy, may be useful for medication-related osteonecrosis of the jaw patients.

13. Effect of Low-Intensity Pulsed Ultrasound (LIPUS) on Remote Bone Marrow in Rats With Healing Socket.

OBJECTIVE: We reported at the previous annual meeting that LIPUS treatment of the molar tooth sockets of retired breeder rats accelerated alveolar bone healing, and that associated humoral effects were seen with elevated blood flow. Namely, LIPUS induced VEGF/angiogenesis along with elevated baseline blood flow rate, which was further associated with a sudden depression of blood flow rate in the socket immediately after cessation of LIPUS treatment. Prior injection with EP4 PGE2 receptor antagonist, but not EP3 antagonist, abolished this LIPUS-induced depression, and topical application of PGE2 to the socket epithelium mimicked the LIPUS-induced depression. In fact, the serum level of PGE2 increased after LIPUS treatment, and significantly increased in the blood flow rate at remote sites on the foot dorsum and tail after 20 minutes. Therefore, in the current study, we examined the tibia bone marrow, which is likely to respond to circulating PGE2. METHODS: Right maxillary first molars were removed from retired female breeder rats in both the LIPUS and the control groups. LIPUS was applied extrabuccally to the socket every 24 hours for 2 weeks starting one day after extraction. Removed bone samples were fixed with 4% formaldehyde to prepare undecalcified frozen sections using Kawamoto's method for immunohistochemical or histochemical staining. Bone marrow samples dissected from the tibia were treated with RNAlater (Ambion) for later RT-PCR analysis. RESULTS AND DISCUSSION: Chemokine receptor CXCR4-positive bone marrow cells increased in the tibia of the LIPUS-treated rat. Together with ubiquitously expressed CXCL12(SDF-1), it is suggested that PGE2 released from the exposed socket is responsible for the recruitment, proliferation and mobilization of the precursors of bone forming cells. LIPUS is thought to exert humoral effects by recruiting bone marrow cells into the healing socket along with VEGF/angiogenesis induced by PGE2.

Increased oxidative stress biomarkers in the saliva of Down syndrome patients.

OBJECTIVE: The DNA oxidation byproduct 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a well-known biomarker used to evaluate oxidative stress. We previously reported that the generation of reactive oxygen species (ROS) is increased in cultured gingival fibroblasts (GF) from patients with Down syndrome (DS). Thus, the aim of this study was to evaluate 8-OHdG as a marker of oxidative stress in saliva of DS patients. MATERIALS AND METHODS: The study group consisted of DS patients (66 patients; age range 1-62 years) and systemically healthy control subjects (71 subjects; age range 4-58 years). Periodontal status was judged based on standard measurements of probing depth (PD) and gingival index (GI). The salivary levels of 8-OHdG were determined using an enzyme-linked immunosorbent assay. RESULTS: The mean of PD and GI values were not significantly different between young (1-12 years) patients with DS (DS-1) and controls (C-1) or between adult (30-62 years) patients with DS (DS-2) and controls (C-2). There were statistically significant positive correlations between the salivary 8-OHdG levels and GI in the DS-1, DS-2 and C-2 groups, but not in the C-1. There were also statistically significant positive correlations between salivary 8-OHdG levels and PD in the DS-2 and C-2 groups, but not in the DS-1 or C-1 groups. The salivary levels of 8-OHdG of DS-1 and DS-2 groups were significantly higher than in the C-l and C-2 groups, respectively. CONCLUSIONS: These results suggest that progressive oxidative stress occurred in DS patients. Oxidative stress may contribute to the clinical features of DS, particularly to the progressive periodontitis characteristic of early ageing.

Reactive oxygen species production in mitochondria of human gingival fibroblast induced by blue light irradiation.

In recent years, it has become well known that the production of reactive oxygen species (ROS) induced by blue-light irradiation causes adverse effects of photo-aging, such as age-related macular degeneration of the retina. Thus, orange-tinted glasses are used to protect the retina during dental treatment involving blue-light irradiation (e.g., dental resin restorations or tooth bleaching treatments). However, there are few studies examining the effects of blue-light irradiation on oral tissue. For the first time, we report that blue-light irradiation by quartz tungsten halogen lamp (QTH) or light-emitting diode (LED) decreased cell proliferation activity of human gingival fibroblasts (HGFs) in a time-dependent manner (<5 min). Additionally, in a morphological study, the cytotoxic effect was observed in the cell organelles, especially the mitochondria. Furthermore, ROS generation induced by the blue-light irradiation was detected in mitochondria of HGFs using fluorimetry. In all analyses, the cytotoxicity was significantly higher after LED irradiation compared with cytotoxicity after QTH irradiation. These results suggest that blue light irradiation, especially by LED light sources used in dental aesthetic treatment, might have adverse effects on human gingival tissue. Hence, this necessitates the development of new dental aesthetic treatment methods and/or techniques to protect HGFs from blue light irradiation during dental therapy.

Dental resin curing blue light induced oxidative stress with reactive oxygen species production.

Dental resin curing blue light has been used in the treatment of tooth bleaching and to restore teeth with resin-based composite fillings. However, there has been little consideration of its effect on oral tissues such as dental pulp and oral mucosa. The aim of this study was to investigate whether dental resin curing blue light irradiation affects the dental pulp, especially the blood vessels that are known as the first target of reactive oxygen species (ROS), which play an important role in vascular reactivity. We found that blue light irradiation increased the level of lipid peroxidation in isolated rat aorta blood vessels by measuring malondialdehyde. Furthermore, cell proliferative activity was decreased in a time-dependent manner and apoptosis of human aorta vascular smooth muscle cells (VSMCs) was induced. These results indicated that (ROS) such as hydrogen peroxide and hydroxyl radicals were generated in VSMCs by irradiation with blue light, and they induced cytotoxicity associated with oxidative stress, which increased lipid peroxidation and apoptosis. In addition, N-acetyl-l-cysteine, which is a typical intracellular antioxidant, protected VSMCs against cytotoxicity associated with oxidative stress. These findings suggested that antioxidants may be used to prevent oxidative stress in dental pulp by repeated and/or multiple treatments with blue light irradiation in future dental treatments.

Assessments of salivary antioxidant activity using electron spin resonance spectroscopy.

OBJECTIVE: In recent years, the function of saliva has been focused on evaluation of general status. The relationship between salivary antioxidant activity and periodontal disease progression is unclear. The aim of this study is to assess the relationship between periodontal disease and salivary antioxidant activity towards various reactive oxygen species (ROS) using electron spin resonance (ESR) technique. METHODS: We demonstrated that whole saliva derived rats or human subjects scavenged ROS such as superoxide (O(2)(·-)) and hydroxyl radical (HO(·)) using ESR spectroscopy with spin trapping agent. In addition, we assessed the relationship between antioxidants activity towards ROS and periodontal index with superoxide dismutase (SOD) activity in human subject saliva. RESULTS: Antioxidant activity towards O(2)(·-) was increased by Porphyromonas gingivalis (P. gingivalis) infection in rat, although antioxidant activity towards HO(·) was not changed. In human, a strong correlation (r = 0.88, p < 0.01) recognized between salivary antioxidant activity towards O(2)(·-) and probing pocket depth (PPD). In addition, the intensity of salivary antioxidant activity depended on SOD activity level. SOD activity was also correlated with PPD. CONCLUSIONS: Rat salivary antioxidant activity towards O(2)(·-) was up-regulated by the inflammatory response caused by P. gingivalis infection. Similar response was recognized in human saliva with periodontal index. Additionally, a linear correlation between antioxidant activity towards O(2)(·-) and SOD activity was verified by ESR technique. Therefore, evaluation of the salivary antioxidant activity towards O(2)(·-) might be an effective parameter for the objective assessment of periodontal disease progression.

Reactive oxygen species generation in gingival fibroblasts of Down syndrome patients detected by electron spin resonance spectroscopy.

Oral manifestations of Down syndrome include high susceptibility to gingival inflammation with early onset and rapidly progressive periodontitis. The influence of reactive oxygen species (ROS) on periodontitis of Down syndrome is unclear. The aim of this study was to characterize ROS formation in Down syndrome-gingival fibroblasts (DS-GF) using electron spin resonance (ESR) spin trapping with 5,5-dimetyl-1-pyrolline-N-oxide (DMPO), and to determine whether ROS generation plays a role in the pathogenesis of periodontitis in Down syndrome patients. We observed formation of the DMPO-OH spin adduct, indicating HO* generation from cultured DS-GF and non-DS-GF. The increased HO* generation in cultured DS-GF was strongly decreased in the presence of the H2O2 scavenger, catalase, or the iron chelator, desferal. This may due to the enzymatic ability of over-expressed CuZn-superoxide dismutase in Down syndrome to catalyze the formation of H2O2 from O2*-, thereby increasing the availability of substrate H2O2 for the iron-dependent generation of HO* via the Fenton reaction, suggesting that HO* generated from DS-GF may be involved in progressive periodontitis of Down syndrome.