Kiyotoshi Inenaga

Association of cortisol and dehydroepiandrosterone sulphate levels in serum with periodontal status in older Japanese adults.

BACKGROUND AND AIM The associations between periodontitis and stress-related steroid hormone levels released by the hypothalamic-pituitary-adrenal axis are poorly understood. In this study, we examined the association between levels of the stress-related steroid hormones cortisol and dehydroepiandrosterone-sulphate (DHEAS) and periodontitis in elderly subjects. METHODS A total of 467 subjects participated in this study. Serum cortisol and DHEAS levels were determined, and a medical questionnaire regarding medical conditions and lifestyle was administered. In addition, clinical examinations including probing depth (PD), bleeding on probing (BOP), and clinical attachment loss (CAL) were conducted. RESULTS The subjects were divided into tertiles on the basis of periodontitis severity. When the analysis was stratified by smoking status, we found that cortisol levels were significantly higher in those with severe CAL among subjects who had never smoked. Furthermore, multiple regression analysis showed that a higher level of cortisol was significantly associated with greater numbers of sites with severe CAL only in those who had never smoked, while a somewhat weaker association was also observed regarding cortisol/DHEAS ratio. In contrast, the level of DHEAS in serum was not associated with periodontitis. CONCLUSION There were significant associations between serum cortisol level, including cortisol/DHEAS ratio, and periodontitis severity in elderly subjects who had never smoked.

Rheological Properties of Human Saliva and Salivary Mucins

Abstract It is unclear which factors in saliva contribute to its rheological properties such as viscosity and spinnbarkeit. In this study, we investigated relationships between the rheological properties (viscosity and spinnbarkeit) and two salivary mucin (MUC5B and MUC7) levels in saliva. Unstimulated (UWS) and chewing-stimulated (CWS) whole saliva were collected from healthy young adults, and the viscosities and spinnbarkeits were measured. The image densities of MUC5B and MUC7 bands in saliva were measured by Stains-All staining after electrophoresis. Both the viscosity and spinnbarkeit of UWS were significantly higher than those of CWS. The viscosities were correlated positively with the spinnbarkeit in UWS, but not in CWS. Image-density analysis of mucins demonstrated that the viscosities increased with the levels of MUC5B and the spinnbarkeit with the levels of MUC7. These results suggest that MUC5B contributes to viscosity and MUC7 to spinnbarkeit.

Differences between Orofacial Inflammation and Cancer Pain

Rat models of orofacial cancer exhibit both allodynia and hyperalgesia; however, it is unclear whether cancer-induced pain is secondary to cancer-induced inflammation. To address this question, we compared the effects of an anti-inflammatory drug, indomethacin, on pain and neurochemical changes in the medullary dorsal horn in orofacial inflammation and cancer models. Daily peripheral administration of indomethacin largely suppressed mechanical allodynia and thermal hyperalgesia in the inflammation model. The same procedure suppressed allodynia and hyperalgesia in the cancer model, but the suppression was weak when compared with that in the inflammation model. In the medullary dorsal horn, calcitonin gene-related peptide and substance P levels were significantly increased in the inflammation model, but did not change in the cancer model. These results suggest that pain in the orofacial cancer model is not significantly mediated by cancer-induced peripheral inflammation, although it may have some involvement.

Galanin inhibits neural activity in the subfornical organ in rat slice preparation

The activation of the subfornical organ (SFO), a circumventricular organ, induces water intake and vasopressin release. Since central administrations of galanin (GAL) suppress water intake and vasopressin release, GAL may inhibit the neural activity of SFO neurons. In the present study, we investigated effects of GAL on the SFO using molecular biological, electrophysiological and anatomical techniques. Reverse transcription-polymerase chain reaction analysis demonstrated the presence in the SFO of rats of the mRNAs for each of the three known GAL receptor subtypes (GalR1, GalR2 and GalR3). In extracellular recordings in SFO slice preparations, GAL dose-dependently inhibited the neural activity of cells from a number of recording sites. Many GAL-sensitive SFO neurons showed excitatory responses to angiotensin II (ANGII). The GalR1 agonist M617 inhibited the activity of SFO neurons, whereas the GalR2 and GalR3 agonist GAL(2-11) had almost no effect. In patch-clamp recordings, GAL induced an outward current in SFO neurons without influencing synaptic currents. An immunoelectron microscopic study revealed the existence of GAL-containing synaptic vesicles in the SFO. These results suggest that the SFO has neural inputs involving GAL. The response to GAL is inhibitory, mediated at least in part by GalR1 and provides a plausible explanation for the opposite effects of ANGII and GAL seen in vivo on water intake and vasopressin release.

Pilocarpine-induced Salivation and Thirst in Conscious Rats

The muscarinic receptor agonist pilocarpine is widely used as a sialogogue. It has been well-established that it also induces water intake in animals. However, the mechanisms underlying the relationships between these events are unknown. To address this problem, we examined water intake and parotid salivary secretion in conscious rats. Intraperitoneally injected pilocarpine increased both water intake and salivary secretion. Intracerebroventricularly injected pilocarpine also induced water intake, but not salivary secretion. Intracerebroventricularly applied atropine, a muscarinic receptor antagonist, suppressed the water intake produced by pilocarpine applied intraperitoneally and intracerebroventricularly. However, it did not affect the salivary secretion induced by pilocarpine applied peripherally. We conclude that peripherally applied pilocarpine affects the parotid glands and the thirst center in the central nervous system, while it may induce salivary secretion mainly via peripheral responses, but water intake mainly via the central nervous system.

Central glial activation mediates cancer-induced pain in a rat facial cancer model

Peripheral and central glial activation plays an important role in development of pain hypersensitivity induced by inflammation and nerve injury. However, the involvement of glial cells in cancer pain is not well understood. The present study evaluated the peripheral and central glial activation and the effect of an inhibitor of glial activation, propentofylline, on pain-related behaviors in a rat facial cancer model of the growth of Walker 256B cells in the unilateral vibrissal pad until days 3-4 post-inoculation. As compared with sham animals, the facial grooming period was prolonged, the withdrawal latency to radiant heat stimulation was shortened, and the withdrawal threshold by von Frey hair stimulation was decreased at the inoculated region, indicating the development of spontaneous pain, thermal hyperalgesia and mechanical allodynia. In immunostainings for Iba1 and glial fibrillary acidic protein (GFAP), although there were no morphological changes of GFAP-immunopositive satellite glial cells in the trigeminal ganglion, Iba1-immunopositive microglia and GFAP-immunopositive astrocytes in the medullary dorsal horn showed large somata with cell proliferation. After the daily i.p. administration of propentofylline beginning pre-inoculation, the central glial activation was attenuated, the prolonged facial grooming was partially suppressed, and the induced allodynia and hyperalgesia from day 2 were prevented, without a change in tumor size. These results suggest that glial activation in the CNS, but not in the peripheral nervous system, mediates the enhancement of spontaneous pain and the development of allodynia and hyperalgesia at an early stage in the facial cancer model.

Dynamic magnetic resonance sialography for patients with xerostomia.

OBJECTIVE The aim was to evaluate the utility of dynamic magnetic resonance (MR) sialographic images as a diagnostic tool for patients with xerostomia as chief complaint. METHODS Various parameters of dynamic MR sialographic images from 40 healthy volunteers were compared with those from 28 patients having dry mouth as chief complaint. Dynamic MR sialographic images were acquired using a 1.5-T full-body MR system and 2-dimensional fast asymmetric spin-echo (2D-FASE) sequences. Acquisition of the optimal section using 2D-FASE sequencing with single-section acquisition of thick sections was repeated every 30 s before and after a few drops of 5% citric acid was placed on the tongue. Student t test was used to examine differences between the healthy volunteer and patient groups regarding the maximum area of salivary gland ducts before and after citric acid stimulation. RESULTS The maximum area of the detectable ducts in the patient group was significantly smaller (P < 0.001), whereas the change in detectable duct area was significantly lower (P < 0.001). CONCLUSIONS This study suggests that dynamic MR sialographic images and data can be useful in the diagnosis of patients with dry mouth as chief complaint.

Transient outward K+ currents in rat dissociated subfornical organ neurones and angiotensin II effects

Although angiotensin II inhibits transient outward K+ currents (IAs) in subfornical organ neurones, there is no evidence concerning which Kv channels are involved. We investigated IA‐generating Kv channels in dissociated rat subfornical organ neurones, using molecular, electrophysiological and pharmacological techniques, and studied the effects of angiotensin II. Conventional RT‐PCR showed the presence of mRNAs for channels of the Kv3.4, Kv1.4 and Kv4 families, which are capable of generating IAs. Tetraethylammonium at 1 mm, which blocks Kv3 channel‐derived currents, and blood‐depressing substance‐I, a Kv3.4‐specific blocker, at 2 μm suppressed the IA‐like component of whole‐cell outward currents in some neurones. 4‐Aminopyridine at 5 mm inhibited IAs in the presence of tetraethylammonium at 1 mm. Cd2+ at 300 μm shifted the activation and inactivation curves of the 4‐aminopyridine‐sensitive and tetraethylammonium‐resistant IAs positively. The tetraethylammonium‐resistant IAs showed fast and slow components during the process of recovery from inactivation, but the slow component was not seen in all neurones. The time constant of the fast recovery component was less than 200 ms, while that of the slow recovery component was around 1 s. Using single‐cell RT‐PCR, mRNAs for Kv4.2 and Kv4.3L were detected frequently, but those for Kv1.4 and Kv3.4 were seen only rarely. Angiotensin II at 30 nm inhibited the fast recovery component of tetraethylammonium‐resistant IAs in many neurones. These results suggest that the fast recovery component of the tetraethylammonium‐resistant IA in subfornical organ neurones depends upon Kv4, and that it can be modulated by angiotensin II.

Endothelin Receptor-mediated Responses in Trigeminal Ganglion Neurons

Recent evidence implicates endothelin in nociception, but it is unclear how endothelin activates trigeminal ganglion (TRG) neurons. In the present study, we investigated the expression of the endothelin receptors ETA and ETB and endothelin-induced responses in rat TRG neurons. Double-immunofluorescence studies demonstrated that ETA and ETB were expressed in TRG neurons and that 26% of ETA- or ETB-expressing neurons expressed both receptors. During whole-cell patch-clamp recording, endothelin-1 enhanced an induced current in response to capsaicin, a TRPV1 agonist, in approximately 20% of dissociated neurons. The enhancement was blocked by the PKC inhibitor chelerythrine and by the ETA antagonist BQ-123, but not by the ETB antagonist BQ-788. Ca2+-imaging showed that endothelin-1 increased the intracellular Ca2+ concentration in more than 20% of the dissociated neurons. Importantly, unlike the effect of endothelin-1 on capsaicin-induced current, the Ca2+ response was largely suppressed by BQ-788 but not by BQ-123. These results suggest that ETA-mediated TRPV1 hyperactivation via PKC activation and ETB-mediated Ca2+ mobilization occurs in different subsets of TRG neurons. These endothelin-induced responses may contribute to the induction of orofacial pain. The ETB-mediated function in TRG neurons is a special feature in the trigeminal system because of no ETB expression in dorsal root ganglion neurons.

Nicotinic receptor subtypes in rat subfornical organ neurons and glial cells.

It is unclear which nicotinic acetylcholine receptor (nAChR) subtypes are involved in the nicotinic activation of cells in the subfornical organ (SFO). We investigated the nAChR subtype using molecular biological, electrophysiological, pharmacological and immunohistochemical techniques. The use of reverse transcription-polymerase chain reaction in rats demonstrated the presence of mRNAs for the alpha2, alpha3, alpha4, alpha6, alpha7, beta2 and beta4 subunits in the SFO. The characteristics and dose-dependency of nicotine-induced inward currents in many dissociated SFO neurons were similar to those induced by acetylcholine in the presence of atropine. The nicotine-induced currents were larger than those induced by cytisine in most responding cells, suggesting the predominance of the beta2- rather than the beta4-containing nAChR. NIC-induced currents were significantly inhibited by dihydro-beta-erythroidine (a relatively selective antagonist for alpha4-containing nAChRs, and a partial antagonist for alpha2 or alpha3) at 300 nM in all responding cells. Additionally, the currents were significantly inhibited by alpha-conotoxin MII at 10 nM (a selective antagonist for alpha3- and/or alpha6-containing nAChRs) in some but not all responding cells. Methyllycaconitine at 10 nM (a selective alpha7-nAChR antagonist) reduced the nicotine-induced current significantly, but to a lesser extent. Fluorescence-labeled alpha-bungarotoxin (a homomeric alpha7 subtype selective binding drug) binding and immunofluorescence for the alpha7 subunit showed that positive images almost overlapped with those immunopositive for an astrocyte marker. These results suggest that the alpha4beta2 subtype is the main functional receptor in SFO neurons while alpha2, alpha3, alpha6, and beta4 subunits have some effect, and homomeric the alpha7 subtype exists in SFO astrocytes.