Kenichi Sasaguri

Expression and Localization of Chromogranin A Gene and Protein in Human Submandibular Gland

Human saliva chromogranin A (CgA) is clinically promising as a psychological stress marker. However, expression of CgA is poorly understood in humans, although salivary gland localization of CgA in other mammals, such as rodents and horses, has been demonstrated. In the present study, we investigated the expression and localization of CgA in the human submandibular gland (HSG) using various methods. CgA was consistently localized in serous and ductal cells in HSG, as detected by immunohistochemistry and in situhybridization. Reactivity was stronger in serous cells than in ductal cells. In addition, strong immunoreactivity for CgA was observed in the saliva matrix of ductal cavities. Western blotting gave one significant immunoreactive band of 68 kDa in the adrenal gland, HSG and saliva. Finally, CgA was detected in secretory granules of serous and ductal cells by immunoelectron microscopy. In conclusion, CgA in humans is produced by HSG and secreted into saliva.

Suppression of Stress-induced nNOS Expression in the Rat Hypothalamus by Biting

Nitric oxide (•NO) modulates the activity of the endocrine system in the behavioral response to stress. The purpose of this study was to investigate the effect of restraining the body of an animal on expression of neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) of the hypothalamus, and the inhibitory effect of para-masticatory activity on restraint-induced nNOS expression. We observed an increase in nNOS mRNA expression and nNOS-positive neurons in the rat hypothalamus after 30 or 60 min of restraint. Biting on a wooden stick during bodily restraint decreased nNOS mRNA expression in the hypothalamus. In addition, the number of nNOS-positive neurons was significantly reduced in the PVN of the hypothalamus. These observations clearly suggest a possible anti-stress effect of the masticatory activity of biting, and this mechanism might be unconsciously in operation during exposure to psychological stressors.

Allowing animals to bite reverses the effects of immobilization stress on hippocampal neurotrophin expression

Acute immobilization stress alters the expression of neurotrophins, including brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), in rat hippocampus. We found that biting may be associated with reduction of systemic stress responses. The purpose of this study was to examine whether neurotrophin expression in rat hippocampus is influenced by biting. Rats were exposed to immobilization stress for 2 h (stress group without biting) or biting for the latter half of 2-hour immobilization stress (biting group). Adrenocorticotropic hormone (ACTH) and corticosterone levels were markedly elevated in the stress group, while the increases in ACHT and corticosterone were suppressed in the biting group. Decreased BDNF mRNA and increased NT-3 mRNA expression in hippocampus were detected on real-time polymerase chain reaction (PCR) in the stress group. The decrease in BDNF mRNA under acute immobilization stress was recovered by biting. In addition, the magnitude of increase in NT-3 mRNA was decreased. No changes in expression of tyrosine receptor kinase B or C, the receptors for BDNF and NT-3, respectively, were observed in this model. These findings suggest that biting influences the alterations in neurotrophin levels induced by acute immobilization stress in rat hippocampus.

Suppression of stress immobilization-induced phosphorylation of ERK 1/2 by biting in the rat hypothalamic paraventricular nucleus

We have previously reported that acute immobilization stress induces Fos protein. Fos protein is generally used as a marker for neuronal activity and has been linked to phosphorylation of extracellular signal-regulated protein kinase 1/2 (pERK1/2), in the hypothalamic paraventricular nucleus (PVN). Biting behavior during the period of stress reduced the expression of Fos protein. The present immunohistochemical study was designed to determine whether acute immobilization stress induces pERK1/2 in the PVN, and whether the stress-induced pERK1/2 was attenuated by simultaneous biting behavior. Acute immobilization stress, in increments of up to 15min, produced detectable amounts of pERK1/2 that were proportional to the interval of stress. Biting during the acute immobilization stress significantly reduced the amount of detectable pERK1/2. These results suggest that biting activity during acute stress inhibits pERK1/2 in this region of the brain. It is feasible that the neuronal cellular response to acute stress is regulated, in some part, by inhibition of pERK1/2 by biting.

CHEWING AMELIORATES STRESS-INDUCED SUPPRESSION OF HIPPOCAMPAL LONG-TERM POTENTIATION

Research has established that severe stress adversely affects hippocampal memory, and chewing has been suggested to restore impaired cognitive functions in the hippocampus. To address how chewing involves stress-attenuated hippocampal memory process, we measured the long-term potentiation (LTP) of hippocampal slices of adult male rats that had experienced restraint stress, including some rats that were allowed to chew a wooden stick during the stress period and other rats that were not. The three experimental conditions were: 1) restraint stress without chewing (ST), 2) restraint stress with chewing (SC), and 3) no treatment (CT). We prepared hippocampal slices and collected trunk blood from all experimental animals. For rats in the two stressed groups, we collected tissue and blood at one of three post-stress time points: immediately after, 24 h after, or 48 h after exposure to the stressor. We found that the magnitude of LTP in both group ST and SC was significantly attenuated immediately after stress exposure. However, within 24 h after the end of the stress period, LTP had returned to the control level in group SC whereas it remained low in group ST. At the same post-stress time point, we found that facilitation of N-methyl-D-aspartate (NMDA) receptors by bath-applied glycine had less effect on the magnitude of LTP in group SC than on group ST, suggesting that most NMDA receptors had already become functionally restored in group SC by that time. Plasma concentration of adrenocorticotropic hormone was significantly elevated only in group ST immediately after exposure to the stressor, reflecting the involvement of chewing in decreasing subsequent corticosterone secretion. Thus, the present study demonstrates that chewing ameliorates the stress-induced impairment of NMDA receptor-mediated LTP, suggesting chewing as a good strategy to cope with severe stress by suppressing excessive endocrine responses.

Submandibular Glands Contribute to Increases in Plasma BDNF Levels

Brain-derived neurotrophic factor (BDNF) promotes survival and differentiation of neural cells in the central and peripheral nervous systems. BDNF has been detected in plasma, but its source has not yet been established. Expression of BDNF mRNA has been identified in the submandibular glands when male rats are exposed to acute immobilization stress. In the present study, we investigated whether plasma BDNF is influenced by the submandibular glands in this model. Acute immobilization stress for 60 min significantly increased the level of plasma BDNF. However, plasma BDNF elevation was markedly suppressed in bilaterally sialoadenectomized rats. There were no significant differences between stressed (60 min) and non-stressed rats with respect to the BDNF mRNA expression in the hippocampus, heart, lung, liver, pancreas, or spleen, as determined by real-time polymerase chain-reaction. These findings suggest that the submandibular glands may be the primary source of plasma BDNF in conditions of acute immobilization stress.

Immobilization stress induces BDNF in rat submandibular glands.

Brain-derived neurotrophic factor (BDNF) promotes survival and differentiation of the cells of the central and peripheral nervous systems. BDNF has been identified in non-neural tissue, including the heart, lung, platelets, lymphocytes, and lacrimal glands. Immobilization stress modifies BDNF mRNA expression in some organs. The present study examines the effect of immobilization stress on BDNF, and its receptor TrkB, in male rat submandibular glands. Increased BDNF mRNA and protein expression were observed in duct cells as a result of immobilization stress, as demonstrated by real-time PCR, Western blot, immunohistochemistry, and analysis by microdissection. TrkB mRNA was not detected in salivary gland tissue, or oral or esophageal mucosa, by RT-PCR. Rat submandibular gland was thus identified as an organ which expresses BDNF. Furthermore, the results of this study suggest that increased salivary BDNF expression occurs following immobilization stress.

Effects of Mandibular Deviation on Brain Activation During Clenching: An fMRI Preliminary Study

Abstract Using functional magnetic resonance imaging (fMRI) in eight healthy human subjects, the present study measured blood oxygenation level-dependent (BOLD) signals during clenching in a malocclusion model, using a custom-made splint that forced the mandible to a retrusive position and a splint of no modification for control, and compared the results to the BOLD signals during the corresponding resting conditions. An individual visual analog scale (VAS) score was also examined during clenching to evaluate the interactions between fMRI data and psychiatric changes. During both clenchings, activations in four brain regions (premotor cortex, prefrontal cortex, sensorimotor cortex, and insula) were seen. However, clenching in the malocclusion model, with psychological discomfort, increased additionally BOLD signals in the anterior cingulate cortex and the amygdala. Furthermore, there was a parallel relationship between BOLD signal intensities and VAS scores in these two regions. The findings may suggest the involvement of clenching with malocclusal conditions in the emotion and/or pain-related neural processing in the brain.

The Use of a BruxChecker in the Evaluation of Different Grinding Patterns During Sleep Bruxism

Abstract A variety of problems involving the masticatory system can be partially attributed to parafunctional habits such as bruxism. These include occlusal trauma, abfractions, tooth migration, as well as temporomandibular dysfunction. Since bruxism is considered a contributing factor to the above-mentioned dental problems, it is essential to consider parafunctional habits in the diagnosis and treatment planning before doing any occlusal reconstruction. However, the problem lies in the lack or absence of a simple device or gauge useful to be able to diagnose and evaluate the occlusal schemes in the patient’s grinding pattern. In this study, we have developed a very simple device (BruxChecker) for evaluating the grinding patterns in sleep bruxism. Using the BruxChecker, it was possible to visualize real or actual interferences during sleep bruxism. Therefore, examination of the grinding pattern using this device is necessary and crucial for making the proper treatment plan for each patient.

Expression of Runx2/Cbfa1/Pebp2αA During Angiogenesis in Postnatal Rodent and Fetal Human Orofacial Tissues†

Transient expression of Runx2 is reported in endothelial cells and vascular smooth muscle cells during vessel formation in skin, stroma of forming bones and developing periodontal ligament, developing skeletal muscle cells, and fat tissue. The data suggest that Runx2 is expressed in a multipotential mesenchymal cell population that gives rise to various osseous and nonosseous cell lineages.