Keiko Unno

Suppressive effect of green tea catechins on morphologic and functional regression of the brain in aged mice with accelerated senescence (SAMP10).

Green tea catechins (GT-catechins) have been reported to have an antioxidative effect. We investigated the effect of long-term GT-catechin intake on aging and oxidative damage using aged mice with accelerated senescence (SAMP10), a model of brain senescence with cerebral atrophy and cognitive dysfunction. Major atrophy was observed in the rhinencephalon, hippocampus and striatum of 12-month-old untreated SAMP10 mice. Similarly, levels of 8-oxodeoxyguanosine (8-oxodG), a marker of oxidative DNA damage, were higher in these parts of the cerebrum than in the cerebral cortex and liver. GT-catechin intake effectively suppressed such atrophy in 12-month-old SAMP10 mice. A preventive effect of GT-catechin intake on oxidative DNA damage was also observed in the rhinencephalon (an area particularly susceptible to atrophy) at 6 months of age, i.e. during the early stages of atrophy. A suppressive effect of GT-catechin intake on cognitive dysfunction, as determined by the learning time needed to acquire an avoidance response and assessments of working memory in a Y-maze, was also found in 12-month-old mice. These results suggest that GT-catechin intake partially improves the morphologic and functional alterations that occur naturally in the brains of aged SAMP10 mice.

Age‐related increase of superoxide generation in the brains of mammals and birds

Oxidative stress, an imbalance between endogenous levels of oxygen radicals and antioxidative defense, increases with aging. However, it is not clear which of these two factors is the more critical. To clarify the production of oxygen radicals increases with age, we examined oxygen radical‐dependent chemiluminescent signals in ex vivo brain slices using a novel photonic imaging method. The chemiluminescent intensity was significantly decreased by the membrane permeable superoxide dismutase (SOD)/catalase mimic, but not by Cu,Zn‐SOD. Inhibitors for complex I, III, and IV of the mitochondrial electron transport chain transiently enhanced the chemiluminescent signal. The superoxide‐dependent chemiluminescent intensity in senescence accelerated mouse (SAM) brain tissues increases with age. Moreover, the slope of the age‐dependent increase was steeper in SAMP10, a strain characterized by a short lifespan and atrophy in the frontal cerebral cortex, than the senescence‐resistant strain SAMR1, which has a longer lifespan. An increase in chemiluminescence with age was also observed in C57/BL6 mice, Wistar rats, and pigeons, although levels of chemiluminescence were lower in the pigeons than murines. The rate of age‐related increases of superoxide‐dependent chemiluminescence was inversely related to the maximum lifespan of the animals. The activity of superoxide dismutase was unchanged during the aging process in the brain. This suggested that superoxide production itself may increase with age. We speculated that reactive oxygen may be a signal to determine the aging process.

Decline in glutathione peroxidase activity is a reason for brain senescence: consumption of green tea catechin prevents the decline in its activity and protein oxidative damage in ageing mouse brain

The accumulation of oxidative damage is believed to contribute to senescence. We have previously found that the consumption of green tea catechins (GT-catechin), which are potent antioxidants, decreases oxidative damage to DNA and improves brain function in aged mice with accelerated senescence (SAMP10 mice). To investigate the mechanisms underlying the beneficial effects of GT-catechin, we measured the activities of antioxidative enzymes in the brains of aged SAMP10 mice. The activity of glutathione peroxidase (GPx), an essential enzyme for reduction of hydrogen and lipid peroxides, was significantly lower in aged mice than in younger ones. However, the decline in activity was prevented in aged mice that had consumed GT-catechin. The increased level of carbonyl proteins, a marker of oxidative damage in proteins, was also significantly reduced in aged mice that had consumed GT-catechin. The activities of superoxide dismutase and catalase were not decreased in aged mice. These results suggest that decreased activity of GPx importantly contributes to brain dysfunction in ageing SAMP10 mice. Furthermore, the intake of GT-catechin protected the decline in GPx activity and age-related oxidative damage in the brain.

Theanine intake improves the shortened lifespan, cognitive dysfunction and behavioural depression that are induced by chronic psychosocial stress in mice

Abstract To evaluate the psychosocial effect on lifespan and cognitive function, this study investigated the effect of confrontational housing on mice because conflict among male mice is a psychosocial stress. In addition, it investigated the anti-stress effect of theanine (γ-glutamylethylamide), an amino acid in tea. Mice were housed under confrontation. That is, two male mice were separately housed in the same cage with a partition for establishing the territorial imperative in each mouse. Then, the partition was removed and mice were co-housed confrontationally (confront-housing) using a model mouse of accelerated-senescence (SAMP10) that exhibited cerebral atrophy and cognitive dysfunction with ageing. It was found that mice began to die earlier under confront-housing than group-housed control mice. Additionally, it was found that cerebral atrophy, learning impairment and behavioural depression were higher in mice under the stressed condition of confront-housing than age-matched mice under group-housing. Furthermore, the level of oxidative damage in cerebral DNA was higher in mice housed confrontationally than group-housed control mice. On the other hand, the consumption of purified theanine (20 μg/ml, 5–6 mg/kg) suppressed the shortened lifespan, cerebral atrophy, learning impairment, behavioural depression and oxidative damage in cerebral DNA. These results suggest that psychosocial stress accelerates age-related alterations such as oxidative damage, lifespan, cognitive dysfunction and behavioural depression. The intake of theanine might be a potential candidate for suppression of disadvantage under psychosocial stress.

Anti-stress effect of theanine on students during pharmacy practice: positive correlation among salivary α-amylase activity, trait anxiety and subjective stress.

PURPOSE Theanine, an amino acid in tea, has significant anti-stress effect on experimental animals under psychosocial stress. Anti-stress effect of theanine on humans was evaluated in 5th-year university students during pharmacy practice. METHOD The study design was a single-blind group comparison and participants (n=20) were randomly assigned to theanine or placebo groups. Theanine or placebo (lactose) tablets (200 mg, twice a day, after breakfast and lunch) were taken from 1 week prior to the pharmacy practice and continued for 10 days in the practice period. To assess the anxiety of the participants, the state-trait anxiety inventory test was carried out before the pharmacy practice. Salivary α-amylase activity (sAA) was measured as a marker of sympathetic nervous system activity. RESULTS In the placebo-group, sAA in the morning (pre-practice sAA) was higher than in theanine-group during the pharmacy practice (p=0.032). Subjective stress was significantly lower in the theanine-group than in the placebo-group (p=0.020). These results suggest that theanine intake had anti-stress effect on students. Furthermore, students with higher pre-practice sAA showed significantly higher trait anxiety in both groups (p=0.015). Similarly, higher pre-practice sAA was correlated to shorter sleeping time in both groups (p=0.41×10(-3)). CONCLUSION Stressful condition increased the level of sAA that was essentially affected by individual trait anxiety. The low levels of pre-practice sAA and subjective stress in the theanine-group suggest that theanine intake suppressed initial stress response of students assigned for a long-term commitment of pharmacy practice.

Endotoxemia-induced cytokine-mediated responses of hippocampal astrocytes transmitted by cells of the brain-immune interface.

Systemic inflammation shifts the brain microenvironment towards a proinflammatory state. However, how peripheral inflammation mediates changes in the brain remains to be clarified. We aimed to identify hippocampal cells and cytokines that respond to endotoxemia. Mice were intraperitoneally injected with lipopolysaccharide (LPS) or saline, and examined 1, 4, and 24 h after injection. Tissue cytokine concentrations in the spleens and hippocampi were determined by multiplex assays. Another group of mice were studied immunohistologically. Fourteen cytokines showed an increased concentration in the spleen, and 10 showed an increase in the hippocampus after LPS injection. Cytokines increased at 4 h (CCL2, CXCL1, CXCL2, and interleukin-6) were expressed by leptomeningeal stromal cells, choroid plexus stromal cells, choroid plexus epithelial cells, and hippocampal vascular endothelial cells, all of which were located at the brain–immune interface. Receptors for these cytokines were expressed by astrocytic endfeet. Cytokines increased at 24 h (CCL11, CXCL10, and granulocyte-colony stimulating factor) were expressed by astrocytes. Cells of the brain–immune interface therefore respond to endotoxemia with cytokine signals earlier than hippocampal parenchymal cells. In the parenchyma, astrocytes play a key role in responding to signals by using endfeet located in close apposition to the interface cells via cytokine receptors.

Ingestion of theanine, an amino acid in tea, suppresses psychosocial stress in mice

•  What is the central question of this study? In the resident‐intruder model of psychosocial stress, the subordinate is aggressively attacked by the dominant, thus the two animals cannot be co‐housed for extended periods. We have developed a novel chronic model of social confrontation, and asked whether the intake of theanine, an amino acid in tea, could suppress stress. •  What is the main finding and its importance? Using a cage partition, we developed a new confrontational housing mouse model of psychosocial stress. Although dominant and subordinate relationships were observed after the removal of the partition, the mice could be co‐housed without physical injury for a long period. Further, we found that the intake of theanine suppressed stress.

Age-related increase of reactive oxygen generation in the brains of mammals and birds: Is reactive oxygen a signaling molecule to determine the aging process and life span?

Since Harman proposed the “free‐radical theory of aging”, oxidative stress has been postulated to be a major causal factor of senescence. The accumulation of oxidative stress‐induced oxidatively modified macromolecules, including protein, DNA and lipid, were found in tissues during the aging process; however, it is not necessarily clear which factor is more critical, an increase in endogenous reactive oxygen and/or a decrease in anti‐oxidative defense, to the age‐related increase in oxidative damage. To clarify the increasing production of reactive oxygen with age, we examined reactive oxygen‐dependent chemiluminescent (CL) signals in ex vivo brain slices prepared from different‐aged animal brains during hypoxia–reoxygenation treatment using a novel photonic imaging method. The CL signal was intensified during reoxygenation. The signals in SAMP10 (short‐life strain) and SAMR1 (control) brain slices increased with aging. The slope of the increase of CL intensity with age in P10 was steeper than in R1. Age‐dependent increase of CL intensity was also observed in C57BL/6 mice, Wistar rats and pigeons; however, superoxide dismutase (SOD) activity in the brain did not change with age. These results suggest that reactive oxygen production itself increased with aging. The rate of age‐related increases of CL intensity was inversely related to the maximum lifespan of animals. We speculate that reactive oxygen might be a signaling molecule and its levels in tissue might determine the aging process and lifespan. Decelerating age‐related increases of reactive oxygen production are expected to be a potent strategy for anti‐aging interventions. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S10–S24.

Protection of brain and pancreas from high-fat diet: effects of catechin and caffeine.

To investigate the effect of a high-fat diet on brain and pancreas functions, we used SAMP10 mice that have characteristics of brain atrophy and cognitive dysfunction with aging. Simultaneously, we investigated the effect of green tea catechin consumption on high-fat diet feeding, because green tea catechin has been reported to improve brain atrophy, brain dysfunction and obesity. The body weight of mice fed a high-fat diet from 2 to 12 months was higher than that of the control, although the calorie intake was not. The high-fat diet also increased insulin secretion; however, the hypersecretion of insulin and obesity were suppressed when mice were fed a high-fat diet with green tea catechin and caffeine. Furthermore, brain atrophy was suppressed and the working memory, tested using Y-maze, improved in mice fed a high-fat diet containing green tea catechin and caffeine. The secretion of insulin might affect both obesity and brain function. A strong correlation was found between working memory and insulin release in mice fed a high-fat diet with green tea catechin and/or caffeine. The results indicate the protective effect of green tea catechin and caffeine on the functions of brain and pancreas in mice fed a high-fat diet.

Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues

In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2)  > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus.