Mu opioid receptor expressing neurons in the rostral ventromedial medulla are the source of mechanical hypersensitivity induced by repeated restraint stress.

Repeated exposure to psychophysical stress often causes an increase in sensitivity and response to pain. This phenomenon is commonly called stress-induced hyperalgesia (SIH). Although psychophysical stress is a well-known risk factor for numerous chronic pain syndromes, the neural mechanism underlying SIH has not yet been elucidated. The rostral ventromedial medulla (RVM) is a key output element of the descending pain modulation system. Descending signals from the RVM have a major impact on spinal nociceptive neurotransmission. In the present study, to clarify changes in the descending pain modulatory system in rats with SIH, we examined the expression of Mu opioid receptor (MOR) mRNA, MeCP2 and global DNA methylation in the RVM after repeated restraint stress for 3 weeks. Additionally, we microinjected neurotoxin dermorphin-SAP into the RVM. The repeated restraint stress for 3 weeks induced mechanical hypersensitivity in the hind paw, a significant increase in the expression of MOR mRNA and MeCP2, and a significant decrease in global DNA methylation in the RVM. The MeCP2 binding to MOR gene promoter in the RVM was significantly decreased in rats with repeated restraint stress. Furthermore, microinjection of dermorphin-SAP into the RVM prevented the mechanical hypersensitivity induced by repeated restraint stress. Although, because of the lack of specific antibody to MOR, we could not show a quantitative analysis in the number of MOR-expressing neurons after the microinjection, these results suggest that MOR-expressing neurons in the RVM induce SIH after repeated restraint stress.

Deficiency of the circadian clock gene Rev-erbα induces mood disorder-like behaviours and dysregulation of the serotonergic system in mice.

Mood disorders such as depression, anxiety, and bipolar disorder are highly associated with disrupted daily rhythms of activity, which are often observed in shift work and sleep disturbance in humans. Recent studies have proposed the REV-ERBα protein as a key circadian nuclear receptor that links behavioural rhythms to mood regulation. However, how the Rev-erbα gene participates in the regulation of mood remains poorly understood. Here, we show that the regulation of the serotonergic (5-HTergic) system, which plays a central role in stress-induced mood behaviours, is markedly disrupted in Rev-erbα-/- mice. Rev-erbα-/- mice exhibit both negative and positive behavioural phenotypes, including anxiety-like and mania-like behaviours, when subjected to a stressful environment. Importantly, Rev-erbα-/- mice show a significant decrease in the expression of a gene that encodes the rate-limiting enzyme of serotonin (5-HT) synthesis in the raphe nuclei (RN). In addition, 5-HT levels in Rev-erbα-/- mice are significantly reduced in the prefrontal cortex, which receives strong inputs from the RN and controls stress-related behaviours. Our findings indicate that Rev-erbα plays an important role in controlling the 5-HTergic system and thus regulates mood and behaviour.

Adverse Effects of Circadian Disorganization on Mood and Molecular Rhythms in the Prefrontal Cortex of Mice.

Disturbance of the daily cycles in sleep and wakefulness induced by conditions such as shift work and jet lag can increase the risk of affective disorders including anxiety and depression. The way such circadian disorganization disrupts the regulation of mood, however, is not well understood. More specifically, the impact of circadian disorganization on the daily rhythms of the neuronal function that controls mood remains unclear. We therefore investigated the effects of circadian disorganization on expression rhythms of clock genes as well as immediate early genes (IEGs) in several mood-controlling regions of the brain. To introduce circadian disorganization of behaviors, we exposed male C57BL/6J mice to chronic reversal of the light-dark cycle and we found a marked negative mood phenotype in these mice. Importantly, the most adverse effect of circadian disorganization on expression rhythms of clock and IEGs was observed in the prefrontal cortex (PFC) when compared to that in other mood-related areas of the brain. Dysregulation of molecular rhythms in the PFC is therefore suggested to be associated with the development of mood disorders in conditions including shift work and jet lag.

Impact of heart-specific disruption of the circadian clock on systemic glucose metabolism in mice.

The daily rhythm of glucose metabolism is governed by the circadian clock, which consists of cell-autonomous clock machineries residing in nearly every tissue in the body. Disruption of these clock machineries either environmentally or genetically induces the dysregulation of glucose metabolism. Although the roles of clock machineries in the regulation of glucose metabolism have been uncovered in major metabolic tissues, such as the pancreas, liver, and skeletal muscle, it remains unknown whether clock function in non-major metabolic tissues also affects systemic glucose metabolism. Here, we tested the hypothesis that disruption of the clock machinery in the heart might also affect systemic glucose metabolism, because heart function is known to be associated with glucose tolerance. We examined glucose and insulin tolerance as well as heart phenotypes in mice with heart-specific deletion of Bmal1, a core clock gene. Bmal1 deletion in the heart not only decreased heart function but also led to systemic insulin resistance. Moreover, hyperglycemia was induced with age. Furthermore, heart-specific Bmal1-deficient mice exhibited decreased insulin-induced phosphorylation of Akt in the liver, thus indicating that Bmal1 deletion in the heart causes hepatic insulin resistance. Our findings revealed an unexpected effect of the function of clock machinery in a non-major metabolic tissue, the heart, on systemic glucose metabolism in mammals.

Anomalous changes in atmospheric radon concentration before and after the 2011 northern Wakayama Earthquake (Mj 5.5).

A significant increase in atmospheric radon concentration was observed in the area around the epicentre before and after the occurrence of the shallow inland earthquake in the northern Wakayama Prefecture on 5 July 2011 (Mj 5.5, depth 7 km) in Japan. The seismic activity in the sampling site was evaluated to identify that this earthquake was the largest near the sampling site during the observation period. To determine whether this was an anomalous change, the atmospheric daily minimum radon concentration measured for a 13-year period was analysed. When the residual radon concentration values without the seasonal radon variation and the linear trend was > 3 standard deviations of the residual radon variation corresponding to the normal period, the values were deemed as anomalous. As a result, an anomalous increase in radon concentration was determined before and after the earthquake. In conclusion, anomalous change related to earthquakes with at least Mj 5.5 can be detected by monitoring atmospheric radon near the epicentre.

Annual variation in the atmospheric radon concentration in Japan.

Anomalous atmospheric variations in radon related to earthquakes have been observed in hourly exhaust-monitoring data from radioisotope institutes in Japan. The extraction of seismic anomalous radon variations would be greatly aided by understanding the normal pattern of variation in radon concentrations. Using atmospheric daily minimum radon concentration data from five sampling sites, we show that a sinusoidal regression curve can be fitted to the data. In addition, we identify areas where the atmospheric radon variation is significantly affected by the variation in atmospheric turbulence and the onshore-offshore pattern of Asian monsoons. Furthermore, by comparing the sinusoidal regression curve for the normal annual (seasonal) variations at the five sites to the sinusoidal regression curve for a previously published dataset of radon values at the five Japanese prefectures, we can estimate the normal annual variation pattern. By fitting sinusoidal regression curves to the previously published dataset containing sites in all Japanese prefectures, we find that 72% of the Japanese prefectures satisfy the requirements of the sinusoidal regression curve pattern. Using the normal annual variation pattern of atmospheric daily minimum radon concentration data, these prefectures are suitable areas for obtaining anomalous radon variations related to earthquakes.

Migration of mouse-induced pluripotent stem cells to glioma-conditioned medium is mediated by tumor-associated specific growth factors.

Neural and mesenchymal stem cells have extensive tropism for malignant glioma. The tumor tropism of induced pluripotent stem (iPS) cells was tested using the Matrigel invasion assay. Mouse iPS cells showed a significant tropism to the conditioned media prepared from six rodent and human glioma cell lines and this tropism to the glioma conditioned media was partially blocked by the neutralizing antibodies for four major tumor-associated growth factors [stem cell factor (SCF), platelet-derived growth factor BB (PDGF-BB), stromal-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF)], which are secreted from the malignant gliomas. The tropism of the iPS cells was enhanced by the growth factors in a concentration-dependent manner from 0.1 to 100 ng/ml. The receptors for those growth factors (c-Kit, ICAM-1, CXCR4 and VEGFR2), measured by reverse transcriptase-polymerase chain reaction, were highly up-regulated in the mouse iPS cells compared to the mouse fibroblasts. The results showed that the specific growth factors secreted from the gliomas strongly attracted the iPS cells. Therefore, gene therapies using iPS cells as vectors to deliver anti-tumor agents are novel strategies for the treatment of malignant gliomas that deeply infiltrate the brain.

Involvement of platelet activation by P2Y12 receptor in the development of transplant arteriosclerosis in mice.

BACKGROUND: Although activated platelets influence inflammation by intraplatelet mediators in transplantation, their mechanism of involvement in the progression of transplant arteriosclerosis has not yet been elucidated. We, therefore, investigated this question using P2Y12 receptor knockout (KO) mice, in which platelets cannot be aggregated by adenosine diphosphate stimulation. METHODS: Carotid arteries from 129X1 mice were orthotopically transplanted into wild-type or KO mice in a minor antigen(s)-mismatched strain combination. No immunosuppression was used. Grafts were harvested at 7, 14, 28, and 56 days after transplantation for morphometry and immunohistology. Fluorescence-activated cell sorting and quantitative real-time reverse-transcriptase polymerase chain reaction were performed at 7 and 14 days after transplantation. RESULTS: The intima/media ratio of grafts in KO mice was significantly reduced compared with wild-type mice at 14, 28, and 56 days after transplantation. Fluorescence-activated cell sorting analysis showed a significant reduction of platelet CD154 expression and platelet-leukocyte aggregates in KO mice at 14 days after transplantation. Additionally, levels of intercellular adhesion molecule-1 and CD40 mRNA, and numbers of intercellular adhesion molecule-1- or CD40-positive cells in the grafts were lower in KO mice at 7 and 14 days after transplantation. These reductions resulted in a significant attenuation of CD45-positive leukocytes adhering to the graft vessel wall in KO mice at 14 days after transplantation. CONCLUSION: Diminished platelet function by P2Y12 receptor deficiency attenuates initiation and strongly inhibits progression of transplant arteriosclerosis in mice by diminishing adhesion molecule expression and leukocyte accumulation in the grafts during the early phase after transplantation.

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells.

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.

Nafamostat attenuated the impairment of fibrinolysis in animal sepsis model by suppressing the increase of plasminogen activator inhibitor type 1.

BACKGROUND: In endotoxemia, plasminogen activator inhibitor-1 (PAI-1) increases and develops clinical symptoms by suppressing fibrinolysis. We analyzed therapeutic advantage of nafamostat, a broad-range protease inhibitor, on fibrinolysis in an animal sepsis model. METHODS: Male Wister rats infused with lipopolysaccharide (LPS) (50 mg/kg) alone or together with nafamostat (0.1 mg/kg/hr) for 4 hours were analyzed. RESULTS: Plasma PAI-1 (4.2: 4.0-5.0 ng/mL, median and interquartile range) increased after LPS infusion (3700: 3400-4000), which was attenuated by nafamostat (2300: 2100-2600, p < 0.05). Fibrin(ogen) degradation products after LPS injection (173: 152-182 microg/mL) were further elevated by nafamostat (205: 205-228, p < 0.05), Nafamostat attenuated polymorphonuclear neutrophils infiltration in the liver, and tended to suppress plasma tumor necrosis factor-alpha levels. Nafamostat did not affect thrombin generation, platelet count, markers of liver and kidney function, and overall mortality. CONCLUSIONS: Nafamostat appeared to improve impaired fibrinolysis by suppressing the increase of PAI-1 in plasma, though it did not largely improve clinical parameters.

Plasminogen activator inhibitor type 1 promotes fibrosarcoma cell migration by modifying cellular attachment to vitronectin via alpha(v)beta(5) integrin.

Both urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1) are associated with a poor prognosis in cancer patients. We demonstrate that PAI-1 inhibits human fibrosarcoma cell (HT-1080) adhesion to vitronectin (Vn) via alpha (v)beta (5) integrin, and stimulates cell migration from Vn toward collagen type IV (Col). The cells attached more strongly to Vn and Col than to fibronectin (Fn), whereas PAI-1 interfered with cell attachment to Vn only. An integrin antagonist, RGD peptide, and anti-alpha (v)beta (5) integrin antibodies, which similarly inhibited cell attachment to Vn, also stimulated cell migration from Vn toward Col. u-PA did not modify cell attachment directly, but reversed the PAI-1-mediated inhibitory effect on cell adhesion to Vn, and its stimulatory effect on cell migration from Vn toward Col. Thus HT-1080 cell migration appears to be modified by u-PA and PAI-1, altering cell adhesion to Vn via alpha (v)beta (5) integrin. This may be related to their tumor-promoting effect.

Sexual dimorphism in endotoxin susceptibility after partial hepatectomy in rats.

BACKGROUND/AIMS: Liver failure due to endotoxemia after hepatectomy is a fatal complication. Little is known regarding the gender influence on this pathophysiological condition. This study was conducted to investigate whether a gender difference exists in the endotoxin susceptibility after hepatectomy. METHODS: Sexually mature male and female rats received an intravenous administration of lipopolysaccharide (LPS), as endotoxin, 48h after a two-thirds hepatectomy. RESULTS: The 24-h survival rate after LPS administration was significantly higher in females (75%) than in males (38%). Ovariectomy reduced the survival rate in females to 44%. Plasma tumor necrosis factor-alpha levels 1h after LPS were significantly elevated in males and ovariectomized females. The inducible nitric oxide synthase (iNOS) gene expression in liver and spleen, and consequent nitric oxide production 3h after LPS were significantly enhanced in males and ovariectomized females when compared to females, in addition to less functional and structural liver damage in females. CONCLUSIONS: Our results indicate a gender difference in the susceptibility to endotoxemia in the early phase after hepatectomy. Female tolerance to these conditions may be mediated by an inhibition of excessive inflammatory response in the liver and the spleen, partially via the suppression of iNOS gene up-regulation.

Essential role of endogenous tissue plasminogen activator through matrix metalloproteinase 9 induction and expression on heparin-produced cerebral hemorrhage after cerebral ischemia in mice.

Cerebral hemorrhage associated with antithrombotic and thrombolytic therapy in acute stroke continues to present a major clinical problem. Rupture of the cerebral microvasculature involves the degradation and remodeling of extracellular matrix. Here we demonstrated that the delayed administration of heparin 3 hours after photothrombotic middle cerebral artery occlusion (MCAO) caused cerebral hemorrhage in wild-type (WT) mice but not in tissue plasminogen activator (tPA)-deficient knockout (KO) mice. Heparin administration increased tPA activity and its mRNA expression at 6 and 12 hours after MCAO in the ischemic hemispheres of WT mice. The expression of tPA was enhanced in microglial cells in the ischemic border zone. We also observed an exacerbation of matrix metalloproteinase (MMP) 9 expression at the mRNA level and its conversion to an active form after heparin administration in the ischemic hemisphere in WT mice but not in tPA KO mice. The increased MMP 9 expression was localized in microglial cells and endothelial cells. These findings suggest that endogenous tPA, through the enhancement of MMP 9 expression and proteolytic activation, plays an essential role in the pathogenesis of heparin-produced cerebral hemorrhage. Targeting tPA, MMP 9, or both may provide a new approach for preventing cerebral hemorrhage associated with antithrombotic therapy for stroke in humans.

Tissue-type plasminogen activator has paradoxical roles in focal cerebral ischemic injury by thrombotic middle cerebral artery occlusion with mild or severe photochemical damage in mice.

The role of endogenous tissue-type plasminogen activator (tPA) in focal cerebral ischemic injury (FCII) after middle cerebral artery occlusion was studied using tPA gene-deficient (KO) mice and their wild-type (WT) littermates. The middle cerebral artery was occluded by thrombi induced by three different intensities of photochemical damage, a method that was newly introduced in mice. In both WT and KO mice, the intensity-dependent increase of FCII size was observed. The FCII size in tPA WT mice was smaller than in KO mice in cases of mild damage, whereas the FCII size was larger in WT mice than in KO mice in cases of severe damage. There was no difference in FCII size between WT and KO mice in cases of moderate damage. The number of microthrombi also increased with damage intensity in both WT and KO mice, but was less in WT mice at all intensities of damage. The results support the validity of the model of thrombotic occlusion by photochemical damage in mice, and suggest that endogenous tPA protects FCII through thrombolytic action on transient occlusion of middle cerebral artery with mild damage, but deteriorates on persistent occlusion with severe damage.