Masato Mitome

Melatonin influences the proliferative and differentiative activity of neural stem cells

Abstract:  Though melatonin has a wide variety of biological functions, its effects on the neural stem cells (NSCs) is still unknown. In this study, we examined the effects of melatonin at either physiological (0.01–10 nm) or pharmacological concentrations (1–100 μm) on the proliferation and neural and astroglial differentiation of NSCs derived from the mouse embryo striatum using an in vitro culture system. We found that melatonin at pharmacological concentrations, but not at physiological concentrations, suppressed epidermal growth factor (EGF)‐stimulated NSC proliferation (increment of viable cells, DNA synthesis and neurosphere formation) in a concentration‐dependent manner. Furthermore, treatment with melatonin at a pharmacological concentration during the proliferation period facilitated 1% FBS‐induced neural differentiation of NSCs without affecting the astroglial differentiation. In contrast, the treatment with melatonin at pharmacological concentrations during the differentiation period decreased the neural differentiation of the NSCs. As with melatonin, MCI‐186, an antioxidant, suppressed EGF‐stimulated NSC proliferation and facilitated the subsequent neural differentiation of NSCs. These results suggest that melatonin exerts potent modulatory effects on NSC functions including the suppression of the proliferation and facilitation of neuronal differentiation, likely via its antioxidant activity. As neurogenesis is thought to play an important role in ameliorating the deficit in neurodegenerative diseases, melatonin might be beneficially used for the treatment diseases such as cerebral infarction.

Mastication influences the survival of newly generated cells in mouse dentate gyrus.

We examined the effects of soft food and tooth loss on neurogenesis in the adult dentate gyrus. Four-week-old mice were subjected to a powder diet for 10 weeks with or without removal of molars. They received a daily injection of bromodeoxyuridine (BrdU) at 14 weeks of age for 12 consecutive days. The number of BrdU-positive cells in the dentate gyrus of these mice did not differ from that of control at 1 day after the last BrdU injection. However, the BrdU-positive cells in these mice showed a larger reduction in number than in control at 5 weeks after the BrdU injection and the ratio of neurons to BrdU-positive cells decreased in the molarless mice. These results suggest that mastication influences the survival of newly generated cells in the adult dentate gyrus.

Circadian rhythm of nitric oxide production in the dorsal region of the suprachiasmatic nucleus in rats

Extracellular concentration of nitrite (NO2-), an oxidized product of nitric oxide (NO), was measured consecutively in the dorsal region of the rat suprachiasmatic nucleus (SCN) by means of in vivo microdialysis. The NO2- concentrations in the dialysates showed robust circadian rhythm under a 12:12 h light/dark cycle and were higher during the dark phase than during the light phase. When the rats were transferred to constant darkness, the 24 h rhythm of NO2- persisted without damping the amplitude. The NO2- level was significantly lowered by an injection of NO synthase inhibitor (NG-monomethyl-L-arginine, 10 mg/kg i.p.). These findings indicate that the daily fluctuation of NO2- in the dorsal region of the SCN, which represents endogenous rhythm of NO, is regulated independently of photic inputs into the SCN and may be related to the circadian clock functions.

Lowered glucose suppressed the proliferation and increased the differentiation of murine neural stem cells in vitro

Cerebral ischemia is known to activate endogenous neural stem cells (NSCs), but its mechanisms remain unknown. Since lowered glucose supply seems to mediate ischemic actions, we examined the effect of low glucose on NSC activities in vitro. Low glucose applied during the proliferation period diminished EGF‐induced proliferation of NSCs without affecting subsequent differentiation, but low glucose directly exposed during the differentiation period facilitated the differentiation of NSCs into neurons and astrocytes. These findings suggest that low glucose facilitated NSC differentiation, but it diminished NSC proliferation. Moreover, the effect of low glucose may be dependent on the timing of application.

Correlative association between circadian expression of mousePer2 gene and the proliferation of the neural stem cells.

We examined the circadian expression of mousePeriod (mPer) genes (mPer1 and mPer2) and the proliferation of the neural stem cells in vitro. The neural stem cells from the ganglionic eminence of embryonic mice were expanded by the neurosphere method and then treated with epidermal growth factor (EGF) to stimulate their mitotic activity. The time courses of the proliferation were examined by WST-8 assay and bromodeoxyuridine (BrdU) incorporation assay and the expression of mPer1 and mPer2 genes was examined by RT-PCR and immunocytochemistry. We have found that EGF treatment elicited the circadian change in both the increase in viable cell number and DNA synthesis activity of the neural stem cells. Also, the gene expression of mPer2, but not mPer1, changed rhythmically with a period of 24 h and correlated negatively with the DNA synthesis activity rhythm. Furthermore, the treatment with an antisense oligonucleotide against mPer2 increased the DNA synthesis activity of the neural stem cells. These results suggest that mPer2 might periodically suppress the proliferation of neural stem cells.

Methamphetamine stimulates the release of neuropeptide Y and noradrenaline from the paraventricular nucleus in rats

Effects of methamphetamine (MAP) on the extracellular neuropeptide Y (NPY) and noradrenaline (NA) levels were examined in the vicinity of the paraventricular nucleus (PVN) of freely moving rats by means of push-pull perfusion. The NA and NPY levels increased significantly in 30-60 min and reached the maximum level in 90-120 min after intraperitoneal administration of MAP. The effects were dose-dependent. The maximum levels were 1.6-fold of the pretreatment level for NPY and 7-fold for NA, respectively, when 5.0 mg/kg b.w. of methamphetamine was administered. It is concluded that MAP stimulates the releases of paraventricular NPY and NA, but the effect is more strong for NA than for NPY.

Independence of feeding-associated circadian rhythm from light conditions and meal intervals in SCN lesioned rats.

Free-running period of the feeding-associated circadian rhythm was assessed in suprachiasmatic nucleus (SCN) lesioned rats under three different conditions using a feeding-fasting (FF) paradigm; restricted feeding (RF) with meal intervals of 24 h (T = 24 h) under 24 h light-dark cycle (LD), RF with T = 24 h under continuous dim light (dim LL), and RF with T = 25 h under dim LL. After the termination of RF, the rats were subjected to FF regimen five times repeatedly, in which food and water were available for 7 days, followed by total food deprivation for 3 days with free-access to water. Free-running period, which was measured with reference to the prefeeding activity peak during food deprivation, was very close to 24 h and was not different under three conditions. It is concluded that the feeding-associated circadian rhythm has a major period close to 24 h, which is not affected either by light conditions nor by meal intervals.

Circadian rhythms of S-IgA and cortisol in whole saliva —Compensatory mechanism of oral immune system for nocturnal fall of saliva secretion—

Abstract Secretory immunoglobulin A (S-IgA) plays the major role for protecting mucosal tissue from infection, and its level in saliva is thought to be the indicator of immune function in the oral cavity. In the present study, we measured the levels of S-IgA, cortisol and total protein in whole saliva as well as flow rate in healthy young adults (n=8) throughout a 24-hr period, and found that salivary S-IgA, cortisol and flow rate fluctuated in a circadian manner with their acrophases (peak time of the rhythms) at 06:50h, 09:04h and 19.01h, respectively ( P l for S-IgA, 8.56ng/m l for cortisol, and 0.47m l /min for the flow rate. The acrophase of S-IgA rhythm corresponded to the nadir of the flow rate rhythm, indicating that these two rhythms are anti-phase to each other. We suggest that the circadian rhythm of S-IgA is not simply subject to daily fluctuation of the volume of salivary fluid that dilutes S-IgA, but is influenced directly or indirectly by central circadian pacemaker, because the ratio of S-IgA to total protein in the morning is more than 3 times higher than that in the evening. The elevation of S-IgA concentration in saliva from midnight to early morning may indicate compensatory mechanism of oral immune system to the fall of saliva secretion during sleep.

Fibroblast growth factor 2 inhibits the expression of stromal cell-derived factor 1α in periodontal ligament cells derived from human permanent teeth in vitro

Although cells derived from periodontal ligament (PDL) tissue are reported to have stem cell-like activity and are speculated to play a crucial role for tissue healing and regeneration after injury or orthodontic treatment, mechanisms regulating their recruitment and activation remain unknown. Recently, stromal cell-derived factor 1α (SDF-1α) has been reported to be important for stem cell homing and recruitment to injured sites. The aim of this study was to evaluate whether fibroblast growth factor 2 (FGF-2) affects the expression of SDF-1α in PDL cells derived from human permanent teeth in vitro. Using real-time PCR, the expression of SDF-1α mRNA in PDL cells was inhibited by treatment with 10 ng/ml FGF-2. When PDL cells were treated with SU5402 (an inhibitor of FGF receptor 1) in combination with FGF-2, the FGF-2-reduced expression of SDF-1α was inhibited. In the presence of the JNK inhibitor SP600125, SDF-1α mRNA in PDL cells was not suppressed by the FGF-2 treatment. Western blot analysis also showed that SDF-1α production was suppressed by treatment with FGF-2, but it recovered with treatment by FGF-2 + SU5402. These findings suggest that SDF-1α from PDL cells plays an important role in the regeneration and homeostasis of periodontal tissues via the recruitment of stem cells.