Effect of Sodium-Glucose Cotransporter-2 Inhibitor Administration on Cardiac Rehabilitation in Patients with Type 2 Diabetes Mellitus with Heart Failure.

Cardiac rehabilitation in patients with diabetes mellitus and heart failure may be affected by anti-diabetic drugs. However, there are few reports on the effects of sodium-glucose cotransporter-2 (SGLT2) inhibitors on cardiac rehabilitation. Thus, we retrospectively investigated the patient backgrounds and effects of cardiac rehabilitation in 44 patients admitted to our hospital with heart failure and pre-existing diabetes mellitus. Our results showed that the patients tended to be older, and those who received SGLT2 inhibitors had lower systolic blood pressure and left ventricular ejection fraction on admission than those who did not. Cardiac rehabilitation significantly improved the Short Physical Performance Battery (SPPB) score in all patients, and there was no significant difference in body mass index or in body weight. There were no significant differences in SPPB score at admission, discharge, or change from admission to discharge with or without SGLT2 inhibitors. These results suggest that SGLT2 inhibitors do not affect the change in SPPB scores. SGLT2 inhibitors may thus be used safely without affecting cardiac rehabilitation while adhering to the necessary safety precautions.

Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways.

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg(-/-) mouse ES cells and human cancer cell lines. Parg(-/-) mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg(-/-) mouse ES cells. Parg(-/-) ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.

Cyclooxygenase-2 activity is important in craniofacial fracture repair.

The aim of this study was to examine the effect of cyclooxygenase (COX)-2 on bone repair after craniofacial fracture in mice. A 4-mm fracture was created in the parietal bone of 8-week-old male COX-2 wild-type (COX-2(+/+)) and knockout (COX-2(-/-)) mice. Ribonucleic acid was extracted from the fractured bone and analysed. For morphological and histological analysis, the mice were killed 8 and 12 weeks after treatment, and sections were prepared. Three-dimensional computed tomography was performed, and the sections were stained with hematoxylin-eosin for histological examination. Expression of COX-2 messenger ribonucleic acid was induced in COX-2(+/+) mice, but not in COX-2(-/-) mice. Ossification at the fracture site was almost complete 12 weeks after fracture in COX-2(+/+) mice. In COX-2(-/-) mice, incomplete union had occurred at the fracture site. In both types of mice, the fracture site contained no cartilaginous tissue, and the callus formed from the periosteal side. These results suggest that COX-2 plays an important role in craniofacial fracture repair and that COX-2-selective non-steroidal anti-inflammatory drugs might interfere with fracture repair of the membranous viscerocranium in the clinical setting.

Electrophysiological identification of the functional presynaptic nerve terminals on an isolated single vasopressin neurone of the rat supraoptic nucleus.

Release of arginine vasopressin (AVP) and oxytocin from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is under the control of glutamate-dependent excitation and GABA-dependent inhibition. The possible role of the synaptic terminals attached to SON neurones has been investigated using whole-cell patch-clamp recording in in vitro rat brain slice preparations. Recent evidence has provided new insights into the repercussions of glial environment modifications on the physiology of MNCs at the synaptic level in the SON. In the present study, excitatory glutamatergic and inhibitory GABAergic synaptic inputs were recorded from an isolated single SON neurone cultured for 12 h, using the whole-cell patch clamp technique. Neurones expressed an AVP-enhanced green fluorescent protein (eGFP) fusion gene in MNCs. In addition, native synaptic terminals attached to a dissociated AVP-eGFP neurone were visualised with synaptic vesicle markers. These results suggest that the function of presynaptic nerve terminals may be evaluated directly in a single AVP-eGFP neurone. These preparations would be helpful in future studies aiming to electrophysiologically distinguish between the functions of synaptic terminals and glial modifications in the SON neurones.

Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse ES cells to DNA damaging agents.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg(+/-) and Parg(-/-)) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg(-/-) embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg(+/+) ES cells. Parg(-/-) ES cells exhibited the same growth rate as Parg(+/+) ES cells in culture. Sensitivity of Parg(-/-) ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (Parg(+/+)) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.

Ghrelin potentiates miniature excitatory postsynaptic currents in supraoptic magnocellular neurones.

Ghrelin is an orexigenic peptide discovered in the stomach as a ligand of the orphan G-protein coupled receptor, and participates in the regulation of growth hormone (GH) release. Previous studies have demonstrated that ghrelin suppressed water intake and stimulated the secretion of arginine vasopressin in rats. We examined the effect of ghrelin on the excitatory synaptic inputs to the magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) using whole-cell patch-clamp recordings in in vitro rat and mouse brain slice preparations. The application of ghrelin (10(-7) approximately 10(-6) m) caused a significant increase in the frequency of the miniature excitatory postsynaptic currents (mEPSCs) in a dose-related manner without affecting the amplitude. The increased frequency of the spontaneous EPSCs persisted in the presence of tetrodotoxin (1 microM). Des-n-octanoyl ghrelin (10(-6) m) did not have a significant effect on the mEPSCs. The ghrelin-induced potentiation of the mEPSCs was significantly suppressed by previous exposure to the transient receptor potential vanilloid (TRPV) blocker, ruthenium red (10 microM) and GH secretagougue type 1a receptor selective antagonist, BIM28163 (10 microM). The effects of ghrelin on the supraoptic MNCs in trpv1 knockout mice were significantly attenuated compared to those in wild-type mice counterparts. These results suggest that ghrelin participates in the regulation of synaptic inputs to the MNCs in the SON via interaction with the GH secretagogue type 1a receptor, and that the TRPV1 channel may be involved in ghrelin-induced potentiation of mEPSCs to the MNCs in the SON.

Response of arginine vasopressin-enhanced green fluorescent protein fusion gene in the hypothalamus of adjuvant-induced arthritic rats.

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.

Specific expression of optically active reporter gene in arginine vasopressin-secreting neurosecretory cells in the hypothalamic-neurohypophyseal system.

The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca(2+) imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion.

Chronic osmotic stimuli increase salusin-beta-like immunoreactivity in the rat hypothalamo-neurohypophyseal system: possible involvement of salusin-beta on [Ca2+]i increase and neurohypophyseal hormone release from the axon terminals.

Salusin-alpha and -beta were recently discovered as bioactive endogenous peptides. In the present study, we investigated the effects of chronic osmotic stimuli on salusin-beta-like immunoreactivity (LI) in the rat hypothalamo-neurohypophyseal system. We examined the effects of salusin-beta on synaptic inputs to the rat magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and neurohypophyseal hormone release from both freshly dissociated SONs and neurohypophyses in rats. Immunohistochemical studies revealed that salusin-beta-LI neurones and fibres were markedly increased in the SON and the magnocellular division of the paraventricular nucleus after chronic osmotic stimuli resulting from salt loading for 5 days and dehydration for 3 days. Salusin-beta-LI fibres and varicosities in the internal zone of the median eminence and the neurohypophysis were also increased after osmotic stimuli. Whole-cell patch-clamp recordings from rat SON slice preparations showed that salusin-beta did not cause significant changes in the excitatory and inhibitory postsynaptic currents of the MNCs. In vitro hormone release studies showed that salusin-beta evoked both arginine vasopressin (AVP) and oxytocin release from the neurohypophysis, but not the SON. In our hands, in the neurohypophysis, a significant release of AVP and oxytocin was observed only at concentrations from 100 nm and above of salusin-beta. Low concentrations below 100 nm were ineffective both on AVP and oxytocin release. We also measured intracellular calcium ([Ca(2+)](i)) increase induced by salusin-beta on freshly-isolated single nerve terminals from the neurohypophysis devoid of pars intermedia. Furthermore, this salusin-beta-induced [Ca(2+)](i) increase was blocked in the presence of high voltage activated Ca(2+)channel blockers. Our results suggest that salusin-beta may be involved in the regulation of body fluid balance by stimulating neurohypophyseal hormone release from nerve endings by an autocrine/paracrine mechanism.

A case of tumoural calcinosis in the temporomandibular joint associated with systemic sclerosis.

Systemic sclerosis (SSc) is a relatively rare condition characterized by the excessive production and deposition of collagen within tissue. This condition is thought to be immunologically mediated and, in addition to its notorious cutaneous manifestations, often involves multiple organs. A case is presented of systemic sclerosis associated with extensive tumoural calcinosis in the temporomandibular joint. There has been no evidence of recurrence or complications during approximately 2 years of follow up, but long-term follow up is essential.

Physiological studies of stress responses in the hypothalamus of vasopressin-enhanced green fluorescent protein transgenic rat.

Arginine vasopressin (AVP) plays an important role in stress-induced activation of the hypothalamic-pituitary adrenal axis. In the present study, AVP-enhanced green fluorescent protein (eGFP) transgenic rats were used to investigate changes in AVP-eGFP expression in the hypothalamic paraventricular nucleus (PVN) and the median eminence (ME) upon exposure to stress conditions. The eGFP fluorescence in the parvocellular division of the PVN (pPVN) was markedly increased 5 days after bilateral adrenalectomy (ADX) and it was colocalised with corticotrophin-releasing hormone-like immunoreactivity in the pPVN. Peripheral administration of dexamethasone completely suppressed the increase of eGFP fluorescence in the pPVN and the external layer of the ME (eME) after bilateral ADX. Significant increases of eGFP fluorescence were observed in the pPVN 6, 12, 24 and 48 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS). In the eME, eGFP fluorescence was significantly increased 48 h after i.p. administration of LPS. By contrast, eGFP fluorescence changed neither in the magnocellular division of the PVN, nor the internal layer of the ME after i.p. administration of LPS. Our results indicate that AVP-eGFP transgenic rats are useful animal model to study dynamic changes of AVP expression in the hypothalamus under stressful conditions.

Effects of the short chain sugar acid 2-buten-4-olide on the hypothalamo-pituitary-adrenal axis in normal and adjuvant-induced arthritic rats.

The effects of intraperitoneal (i.p.) administration of 2-buten-4-olide (2-B4O), an endogenous sugar acid, on the hypothalamo-adenohypophysial system were examined in Lewis rats that were normal and in adjuvant-induced arthritic (AA) rats. In comparison with vehicle-treated rats, the plasma corticosterone and c-fos mRNA levels in the paraventricular nucleus (PVN) of normal rats increased significantly after i.p. administration of 2-B4O. Dual immunostaining revealed that almost all corticotrophin-releasing factor (CRF)-immunopositive neurones in the parvocellular division of the PVN exhibited Fos-like immunoreactivity (LI) 120 min after i.p. administration of 2-B4O (100 mg/kg). In the AA rats, repeated i.p. administration of 2-B4O (100 mg/kg) after immunisation significantly suppressed the expression of clinical symptoms and significantly increased plasma concentrations of corticosterone. Further, repeated i.p. administration of 2-B4O significantly increased CRF mRNA levels in the PVN and pro-opiomelanocortin mRNA levels in the anterior pituitary; however, they did not change arginine vasopressin mRNA levels in the parvocellular division of the PVN. These results suggest that i.p. administration of 2-B4O activates the hypothalamo-pituitary-adrenal (HPA) axis via the activation of CRF neurones in the PVN, and the activation of the HPA axis by i.p. administration of 2-B4O may be associated with the inhibition of AA in rats.

Exaggerated response of arginine vasopressin-enhanced green fluorescent protein fusion gene to salt loading without disturbance of body fluid homeostasis in rats.

We examined the effects of chronic salt loading on the hypothalamic expressions of the enhanced green fluorescent protein (eGFP), arginine vasopressin (AVP) and oxytocin (OXT) genes in AVP-eGFP transgenic rats that expressed eGFP in the hypothalamic AVP-containing neurones. In these rats, salt loading for 5 days caused a marked increase of the eGFP fluorescence in the magnocellular divisions of the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the internal layer of the median eminence. Expression of the eGFP gene was increased seven- to eight-fold in the PVN and SON of salt-loaded rats in comparison with euhydrated rats. By contrast, none of these changes were observed in the suprachiasmatic nucleus. The expression of the AVP and OXT genes was increased 1.5- to two-fold in the PVN and SON of salt-loaded nontransgenic (control) and transgenic rats. There were no differences in the expression levels of the AVP and OXT genes in the PVN and SON between nontransgenic (control) and transgenic animals under normal conditions and after salt loading. In the posterior pituitary gland, the intensity of the eGFP fluorescence did not change after salt loading for 5 days, but increased after 10 days of salt loading. Upon salt loading, significant increases in the plasma AVP concentrations, plasma osmolality and plasma Na+ were observed. Furthermore, there were no significant differences in changes of water intake, food intake, urine volume, urine osmolality, urine Na+ concentrations, and the body weights in both models under normal or salt-loaded conditions. Our results show that the response of the AVP-eGFP fusion gene to chronic salt loading is exaggerated, and humoral responses such as AVP and OXT and the body fluid homeostasis are maintained in AVP-eGFP transgenic rats. The AVP-eGFP transgenic rat gives us a new opportunity to study the dynamics of the AVP system in vivo.

Prolactin-releasing peptide is a potent mediator of stress responses in the brain through the hypothalamic paraventricular nucleus.

The effects of i.c.v. administration of prolactin-releasing peptide on neurons in the paraventricular nucleus of rats and plasma corticosterone levels were examined by measuring changes in Fos-like immunoreactivity, c-fos mRNA using in situ hybridization histochemistry, and plasma corticosterone using a specific radioimmunoassay. Approximately 80% of corticotropin-releasing hormone immunoreactive cells exhibited Fos-like immunoreactivity in the parvocellular division of the paraventricular nucleus 90 min after i.c.v. administration of prolactin-releasing peptide. The greatest induction of the c-fos mRNA expression in the paraventricular nucleus was observed 30 min after administration of prolactin-releasing peptide, and occurred in a dose-related manner. Plasma corticosterone levels were also significantly increased 30 min after administration of prolactin-releasing peptide. Next, the effects of restraint stress, nociceptive stimulus and acute inflammatory stress on the expression of the prolactin-releasing peptide mRNA in the dorsomedial hypothalamic nucleus, nucleus of the solitary tract and ventrolateral medulla were examined using in situ hybridization histochemistry for prolactin-releasing peptide mRNA. Restraint stress and acute inflammatory stress upregulated the prolactin-releasing peptide mRNA expression in the nucleus of the solitary tract and ventrolateral medulla. Nociceptive stimulus upregulated the prolactin-releasing peptide mRNA expression in the ventrolateral medulla. Finally, we observed that pretreatment (i.c.v. administration) with an anti-prolactin-releasing peptide antibody significantly attenuated nociceptive stimulus-induced c-fos mRNA expression in the paraventricular nucleus. These results suggest that prolactin-releasing peptide is a potent and important mediator of the stress response in the brain through the hypothalamic paraventricular nucleus.

Cannabinoids modulate synaptic activity in the rat supraoptic nucleus.

In the present study, we investigated the effects of the cannabinoid receptor agonist CP55,940 on excitatory and inhibitory synaptic transmission in the rat supraoptic nucleus. Whole-cell patch clamp recordings were performed on supraoptic neurones in in vitro brain slice preparations. CP55,940 significantly reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents in a concentration-dependent manner. These changes were potently reversed by the CB1 receptor antagonist AM251. The results indicate that cannabinoids modulate the activity of magnocellular neurosecretory neurones by presynaptic inhibition of both excitatory and inhibitory synaptic transmission.

Expression of immediate early genes and vasopressin heteronuclear RNA in the paraventricular and supraoptic nuclei of rats after acute osmotic stimulus.

Monitoring the expression of immediate early genes (IEGs) is useful for following stress-induced cellular responses in the neuroendocrine system. We have examined the transcriptional activities of four IEGs (c-fos, junB, NGFI-A and NGFI-B) and of the arginine vasopressin (AVP) gene in the hypothalamic paraventicular (PVN) and supraoptic nuclei (SON) of rats after acute osmotic stimuli, using in situ hybridization histochemistry. After intraperitoneal (i.p.) administration of hypertonic saline (2% body weight, 900 mOsm/kg), the expression levels of all IEG mRNAs were increased significantly both in the PVN and SON at as early as 10 min, peaked at 30 min and remained elevated until 60 min. The expression of AVP heteronuclear (hn)RNA also peaked at 30 min, and remained elevated until 180 min. Thirty min after i.p. administration of hypertonic saline (600 mOsm/kg), the expression levels of all IEG mRNAs in the PVN and SON were significantly increased in comparison with those after i.p. administration of isotonic saline (290 mOsm/kg). Regression analysis revealed that expression levels of the IEG mRNAs and AVP hnRNA were positively correlated with the plasma concentration of sodium, and the rates of increase of the expression levels of all IEG mRNAs were similar. The expression levels of all IEG mRNAs examined are useful markers for following the changes of the AVP gene transcription in the PVN and SON after acute osmotic stimuli in rats.

Changes in the awareness of oral health among new students newly enrolled at the University of Tokyo over the past 15 years.

OBJECTIVE: The aim of this study was to examine changes in awareness of oral health among Japanese university students. METHODS: Between 1990 and 2004, a total of 51,650 students newly enrolled at the University of Tokyo responded to an annual written questionnaire on oral health. RESULTS: (i) Approximately 60% of the students brushed their teeth twice a day. Female students brushed more frequently than male students. (ii) The percentage of students who brushed for 2-3 min per time decreased, while the percentage who brushed four or more minutes increased. (iii) The number of students who had learned how to brush properly increased. This trend was particularly clear-cut among male students, although the proportion of female students who had learned to brush properly remained higher than that of male students. (iv) The percentage of female students who sought treatment for malocclusion was higher than that of male students. The percentage of students who underwent orthodontic treatment increased from 11.6 to 19.7%. The percentage of female students who received orthodontic treatment was approximately twofold that of male students. (v) The percentage of students who had temporomandibular disorders was 0.7% in males and 1.5% in females. (vi) More than 40% of the students had periodontal diseases, with a higher prevalence among male students than female students. (vii) Approximately 20% of the students wanted to consult our service centre. CONCLUSIONS: The awareness of oral health among new undergraduates at the University of Tokyo has improved over the past 15 years.