Relationship between Corticospinal Excitability While Gazing at the Mirror and Motor Imagery Ability.

Mirror therapy (MT) helps stroke survivors recover motor function. Previous studies have reported that an individual's motor imagery ability is related to the areas of brain activity during motor imagery and the effectiveness of motor imagery training. However, the relationship between MT and motor imagery ability and between corticospinal tract excitability during mirror gazing, an important component of MT, and motor imagery ability is unclear. This study determined whether the motor-evoked potential (MEP) amplitude while gazing at the mirror relates to participants' motor imagery abilities. Twenty-four healthy right-handed adults (seven males) were recruited. Transcranial magnetic stimulation was performed while gazing at the mirror, and MEP of the first dorsal interosseous muscle of the right hand were measured. Motor imagery ability was measured using the Kinesthetic and Visual Imagery Questionnaire (KVIQ), which assesses the vividness of motor imagery ability. Additionally, a mental chronometry (MC) task was used to assess time aspects. The results showed a significant moderate correlation between changes in MEP amplitude values while gazing at the mirror, as compared with resting conditions, and assessment scores of KVIQ. This study shows that corticospinal excitability because of mirror gazing may be related to the vividness of motor imagery ability.

Difference between the Effects of Peripheral Sensory Nerve Electrical Stimulation on the Excitability of the Primary Motor Cortex: Examination of the Combinations of Stimulus Frequency and Duration.

Peripheral sensory nerve electrical stimulation (PES) excites the primary motor cortex and is expected to improve motor dysfunction post-stroke. However, previous studies have reported a variety of stimulus frequencies and stimulus duration settings, and the effects of these different combinations on primary motor cortex excitability are not clear. We aimed to clarify the effects of different combinations of stimulus frequency and stimulus duration of PES on the excitation of primary motor cortex. Twenty-one healthy individuals (aged > 18 years, right-handed, and without a history of neurological or orthopedic disorders) were included. Each participant experienced three different stimulation frequencies (1, 10 and 50 Hz) and durations (20, 40 and 60 min). Motor-evoked potentials (MEPs) were recorded pre- and post-PES. The outcome measure was the change in primary motor cortex excitability using the MEP ratio. We used a D-optimal design of experiments and response surface analysis to define the optimal combination within nine different settings inducing more satisfying responses. The combination of stimulation frequency and stimulation time that maximized the desirability value was 10 Hz and 40 min, respectively. The results of this study may provide fundamental data for more minimally invasive and effective implementation of PES in patients with stroke.

Insomnia and depressive behavior of MyD88-deficient mice: Relationships with altered microglial functions.

Myeloid differentiation primary response gene 88 (MyD88) is essential for microglial activation. Despite the significant role of microglia in regulating sleep homeostasis, the contribution of MyD88 to sleep is yet to be determined. To address this, we performed electroencephalographic and electromyographic recordings on MyD88-KO mice and wild-type mice to investigate their sleep/wake cycles. In the daytime, MyD88-KO mice exhibited prolonged wakefulness and shorter non-rapid eye movement sleep duration. Tail suspension and sucrose preference tests revealed that MyD88-KO mice displayed a depressive-like phenotype. We determined monoamines in the prefrontal cortex (PFC) using high-performance liquid chromatography and observed a decreased content of serotonin in the PFC of MyD88-KO mice. Flow cytometry revealed that CD11b, CD45, and F4/80 expressions were elevated at Zeitgeber time (ZT) 1 compared to at ZT13 only in wild-type mice. Furthermore, MFG-E8 and C1qB-tagged synapses were enhanced at ZT1 in the PFC of wild-type mice but not in MyD88-KO mice. Primary cultured microglia from MyD88-KO mice revealed decreased phagocytic ability. These findings indicate that genetic deletion of MyD88 induces insomnia and depressive behavior, at least in part, by affecting microglial homeostasis functions and lowering the serotonergic neuronal output.

Effects of occupational therapy on improvements in the handwriting ability of the adult non-dominant hand: An exploratory randomised controlled trial.

INTRODUCTION: Occupational therapy often involves handwriting acquisition practices that include the non-dominant hand when improvements in the dominant hand function are not possible because of trauma or stroke. This study explored whether character tracing and using a pegboard can effectively improve the handwriting of the non-dominant hand. METHODS: A randomised controlled trial involving 60 healthy university students aged ≥18 years was conducted. Participants were randomly assigned to the writing group, peg group or control group. The character recognition rate was evaluated by computer software. Furthermore, character quality and writing speed were evaluated by humans using global legibility scales. Evaluations were performed before the intervention (baseline) and on days 5 and 10 of the intervention. Using the non-dominant hand, the writing group traced characters on paper with a ballpoint pen, and the peg group used a pegboard for 15 min/day for 10 days. RESULTS: Compared with the peg and control groups, the writing group showed significant improvements in the character recognition rate and global legibility scale score. However, the global legibility scale score did not improve to the same level as that achieved with the dominant hand. None of the evaluation scores of the peg group showed significant improvements compared with those of the control group. There were no significant differences in improvements in the writing speed of the writing and peg groups compared with the control group. CONCLUSION: Tracing characters can improve the handwriting ability of the non-dominant hand, but using a pegboard may be less effective. Future research is needed to examine how much practice is necessary to improve the handwriting ability of the non-dominant hand sufficiently.

Angiotensin II and Amyloid-ß Synergistically Induce Brain Vascular Smooth Muscle Cell Senescence.

BACKGROUND: Amyloid-ß (Aß) induces cerebrovascular damage and is reported to stimulate endothelial cell senescence. We previously demonstrated that angiotensin II (Ang II)-promoted vascular senescence. We examined the possible cross-talk between Ang II and Aß in regulating brain vascular smooth muscle cell (BVSMC) senescence. METHODS: BVSMCs were prepared from adult male mice and stimulated with Ang II (0, 0.1, 1, 10, and 100 nmol/l) and/or Aß 1-40 (0, 0.1, 0.3, 0.5, 1, 3, and 5 µmol/l) for the indicated times. Cellular senescence was evaluated by senescence-associated ß-galactosidase staining. RESULTS: Treatment with Ang II (100 nmol/l) or Aß (1 µmol/l) at a higher dose increased senescent cells compared with control at 6 days. Treatment with Ang II (10 nmol/l) or Aß (0.5 µmol/l) at a lower dose had no effect on senescence whereas a combined treatment with lower doses of Ang II and Aß significantly enhanced senescent cells. This senescence enhanced by lower dose combination was markedly blocked by valsartan (Ang II type 1 receptor inhibitor) or TAK-242 (Aß receptor TLR4 inhibitor) treatment. Moreover, lower dose combination caused increases in superoxide anion levels and p-ERK expression for 2 days, NF-κB activity, p-IκB, p-IKKα/ß, p16 and p53 expression for 4 days, and an obvious decrease in pRb expression. These changes by lower dose combination, except in p-IκB expression and NF-κB activity, were significantly inhibited by pretreatment with U0126 (ERK inhibitor). CONCLUSIONS: Ang II and Aß synergistically promoted BVSMC senescence at least due to enhancement of the p-ERK-p16-pRb signaling pathway, oxidative stress, and NF-κB/IκB activity.

Cerebral Hemodynamics During a Cognitive-Motor Task Using the Limbs.

Background: Antagonistic tasks are cognitive-motor task trainings. Intervention programs involving antagonistic exercise tasks are being employed to help prevent falls and reduce the need for nursing care in older populations. Meanwhile, the effects of such tasks on blood flow in the brain remain obscure. This study aimed to clarify the effects of antagonistic tasks on prefrontal cortical cerebral hemodynamics. Materials and Methods: We assessed 13 healthy adults (two men, 11 women; mean age, 21.4 ± 1.0 years). Participants imitated each of the antagonistic tasks presented on a PC monitor placed at a 120-mm viewing distance. All participants performed six tasks, consisting of upper-limb tasks (non-antagonism, simple antagonism, and complex antagonism) and upper- and lower-limb tasks (tasks combining lower-limb opening and closing movements with each upper-limb task). We used near-infrared spectroscopy (NIRS) to measure cerebral blood flow dynamics, with oxygenated hemoglobin (Oxy-Hb) concentration changes as the main outcome. A 10-channel probe was placed on the participants' forehead, focusing on the prefrontal cortex. We first obtained a baseline NIRS measurement for 10 s; the participants then imitated the task presented on the PC monitor for 90 s. We measured the number of errors and the subjective difficulty of each task. Results: The increase in prefrontal cortex Oxy-Hb concentration was significantly higher in the complex antagonist conditions than in the non-antagonistic and simple antagonistic conditions. There were no significant prefrontal cortex Oxy-Hb differences between the upper limb and upper- and lower-limb conditions (increasing number of motor limbs). Conclusions: The study findings support that an increase in finger-shaped complexity has a greater effect on cerebral blood flow dynamics in the prefrontal cortex than does an increase in the number of motor limbs involved in the task.

Temporal expression profiling of DAMPs-related genes revealed the biphasic post-ischemic inflammation in the experimental stroke model.

The neuroinflammation in the ischemic brain could occur as sterile inflammation in response to damage-associated molecular patterns (DAMPs). However, its long-term dynamic transcriptional changes remain poorly understood. It is also unknown whether this neuroinflammation contributes to the recovery or just deteriorates the outcome. The purpose of this study is to characterize the temporal transcriptional changes in the post-stroke brain focusing on DAMPs-related genes by RNA-sequencing during the period of 28 days. We conducted the RNA-sequencing on day 1, 3, 7, 14, 28 post-stroke in the mouse photothrombosis model. The gross morphological observation showed the ischemic lesion on the ipsilateral cortex turned into a scar with the clearance of cellular debris by day 28. The transcriptome analyses indicated that post-stroke period of 28 days was classified into four categories (I Baseline, II Acute, III Sub-acute-#1, IV Sub-acute-#2 phase). During this period, the well-known genes for DAMPs, receptors, downstream cascades, pro-inflammatory cytokines, and phagocytosis were transcriptionally increased. The gene ontology (GO) analysis of biological process indicated that differentially expressed genes (DEGs) are genetically programmed to achieve immune and inflammatory pathways. Interestingly, we found the biphasic induction of various genes, including DAMPs and pro-inflammatory factors, peaking at acute and sub-acute phases. At the sub-acute phase, we also observed the induction of genes for phagocytosis as well as regulatory and growth factors. Further, we found the activation of CREB (cAMP-response element binding protein), one of the key players for neuronal plasticity, in peri-ischemic neurons by immunohistochemistry at this phase. Taken together, these findings raise the possibility the recurrent inflammation occurs at the sub-acute phase in the post-stroke brain, which could be involved in the debris clearance as well as neural reorganization.

Deterioration of cognitive function after transient cerebral ischemia with amyloid-ß infusion-possible amelioration of cognitive function by AT2 receptor activation.

BACKGROUND: To promote understanding of the pathogenesis of cognitive impairment or dementia, we explored the potential interaction between transient cerebral ischemia and amyloid-ß (Aß) infusion in mediating cognitive decline and examined the possible ameliorative effect of angiotensin II type 2 (AT2) receptor activation in vascular smooth muscle cells (VSMC) on this cognitive deficit. METHODS: Adult male wild-type mice (WT) and mice with VSMC-specific AT2 receptor overexpression (smAT2) were subjected to intracerebroventricular (ICV) injection of Aß1-40. Transient cerebral ischemia was induced by 15 min of bilateral common carotid artery occlusion (BCCAO) 24 h after Aß injection. RESULTS: Aß injection in WT induced a cognitive decline, whereas BCCAO did not cause a significant cognitive deficit. In contrast, WT with BCCAO following Aß injection exhibited more marked cognitive decline compared to Aß injection alone, in concert with increases in superoxide anion production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and expression of p22phox, p40phox, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1ß in the hippocampus, and upregulation of RAGE (receptor for advanced glycation end product), an Aß transporter. BCCAO following Aß injection further enhanced neuronal pyknosis in the hippocampus, compared with BCCAO or Aß injection alone. In contrast, smAT2 did not show a cognitive decline, increase in oxidative stress, inflammation, and RAGE level or neuronal pyknosis, which were induced by BCCAO with/without Aß injection in WT. CONCLUSIONS: Transient cerebral ischemia might worsen Aß infusion-mediated cognitive decline and vice versa, with possible involvement of amplified oxidative stress and inflammation and impairment of the RAGE-mediated Aß clearance system, contributing to exaggerated neuronal degeneration. AT2 receptor activation in VSMC could play an inhibitory role in this cognitive deficit.

AT2 receptor stimulation inhibits phosphate-induced vascular calcification.

Vascular calcification is a common finding in atherosclerosis and in patients with chronic kidney disease. The renin-angiotensin system plays a role in the pathogenesis of cardiovascular remodeling. Here, we examined the hypothesis that angiotensin II type 2 receptor (AT2) stimulation has inhibitory effects on phosphate-induced vascular calcification. In vivo, calcification of the thoracic aorta induced by an adenine and high-phosphate diet was markedly attenuated in smooth muscle cell-specific AT2-overexpressing mice (smAT2-Tg) compared with wild-type and AT2-knockout mice (AT2KO). Similarly, mRNA levels of relevant osteogenic and vascular smooth muscle cell marker genes were unchanged in smAT2-Tg mice, while their expression was significantly altered in wild-type mice in response to high dietary phosphate. Ex vivo, sections of thoracic aorta were cultured in media supplemented with inorganic phosphate. Aortic rings from smAT2-Tg mice showed less vascular calcification compared with those from wild-type mice. In vitro, calcium deposition induced by high-phosphate media was markedly attenuated in primary vascular smooth muscle cells derived from smAT2-Tg mice compared with the two other mouse groups. To assess the underlying mechanism, we investigated the effect of PPAR-γ, which we previously reported as one of the possible downstream effectors of AT2 stimulation. Treatment with a PPAR-γ antagonist attenuated the inhibitory effects on vascular calcification observed in smAT2-Tg mice fed an adenine and high-phosphate diet. Our results suggest that AT2 activation represents an endogenous protective pathway against vascular calcification. Its stimulation may efficiently reduce adverse cardiovascular events in patients with chronic kidney disease.

Deletion of interferon-regulatory factor-1 results in cognitive impairment.

Interferon-regulatory factor (IRF)-1-dependent genes in neurons play a role in ischemic neuronal death; however, the roles of IRF-1 in dementia are not well investigated. Therefore, we assessed the effect of IRF-1 on cognitive function using a vascular cognitive impairment mouse model created by chronic cerebral hypoperfusion. Male 10-week-old C57BL/6 (wild-type; WT) and IRF-1-knockout (IRF-1KO) mice were used in this study. A chronic cerebral hypoperfusion mouse model was generated by bilateral common carotid artery stenosis (BCAS) treatment. After 6 weeks of BCAS, the mice were subjected to the Morris water maze test five times a day for 5 days. In the Morris water maze task, escape latency was significantly prolonged in sham-operated IRF-1KO mice compared with sham-operated WT mice. However, BCAS treatment cancelled such difference in spatial learning between WT and IRF-1KO mice. BCAS treatment decreased CBF, but no significant difference was observed between the two strains after BCAS. Sham-operated IRF-1KO mice showed a decrease in mRNA expression of caspase-1 and an increase in IRF-2 expression in the hippocampus. Expression of angiotensin II type 2 (AT2) receptor, which induces better cognitive function, is regulated by IRF-1; however, no obvious difference in AT2 receptor expression was observed between the two strains even after BCAS. These results suggest that IRF-1 has a protective effect on cognitive decline in a normal condition; however, there was no obvious effect on cognition after chronic cerebral hypoperfusion treatment.

Attenuation of stroke damage by angiotensin II type 2 receptor stimulation via peroxisome proliferator-activated receptor-gamma activation.

The brain renin-angiotensin system plays a crucial role in ischemic stroke. It is known that stimulation of the angiotensin II type 2 (AT2) receptor protects against ischemic brain injury. We recently demonstrated that AT2 receptor stimulation by compound 21 (C21), a direct AT2 receptor agonist, inhibited vascular intimal proliferation with activation of peroxisome proliferator-activated receptor-gamma (PPAR-γ). However, whether direct AT2 receptor stimulation protects against ischemic brain injury via PPAR-γ activation is still unknown. 8-week-old male C57BL/6 J mice were subjected to middle cerebral artery (MCA) occlusion. 2 weeks before MCA occlusion, they were administered C21 with or without GW9662, a PPAR-γ antagonist. Neurologic deficit, ischemic size, superoxide anion, superoxide dismutase (SOD) activity, expression of NADPH subunits and blood brain barrier (BBB) stabilization were assessed 24 h after MCA occlusion. Cerebral blood flow (CBF) was measured in the core and periphery of the MCA territory before, immediately after, 1 h and 24 h after MCA occlusion. Treatment with C21 markedly decreased the neurologic deficit and ischemic size with an increase in CBF, SOD activity and BBB stabilization genes compared with the non-treated group. Co-administration of GW9662 partially attenuated this protective effect of C21 on neurologic deficit and ischemic size via an increase in superoxide anion production and a decrease of SOD activity and BBB stabilization genes, while GW9662 treatment alone had no significant effect on neurologic deficit and ischemic size. These results suggest that direct AT2 receptor stimulation has a preventive effect on stroke-induced brain injury partly due to activation of PPAR-γ.

Recognition of early stage thigmotaxis in Morris water maze test with convolutional neural network.

The Morris water maze test (MWM) is a useful tool to evaluate rodents' spatial learning and memory, but the outcome is susceptible to various experimental conditions. Thigmotaxis is a commonly observed behavioral pattern which is thought to be related to anxiety or fear. This behavior is associated with prolonged escape latency, but the impact of its frequency in the early stage on the final outcome is not clearly understood. We analyzed swim path trajectories in male C57BL/6 mice with or without bilateral common carotid artery stenosis (BCAS) treatment. There was no significant difference in the frequencies of particular types of trajectories according to ischemic brain surgery. The mouse groups with thigmotaxis showed significantly prolonged escape latency and lower cognitive score on day 5 compared to those without thigmotaxis. As the next step, we made a convolutional neural network (CNN) model to recognize the swim path trajectories. Our model could distinguish thigmotaxis from other trajectories with 96% accuracy and specificity as high as 0.98. These results suggest that thigmotaxis in the early training stage is a predictive factor for impaired performance in MWM, and machine learning can detect such behavior easily and automatically.

Beneficial Effect of Mas Receptor Deficiency on Vascular Cognitive Impairment in the Presence of Angiotensin II Type 2 Receptor.

BACKGROUND: The classical renin-angiotensin system is known as the angiotensin (Ang)-converting enzyme/Ang II/Ang type 1 receptor axis, which induces various organ damage including cognitive decline. The angiotensin-converting enzyme 2/Ang-(1-7)/Mas axis is known to exert antagonistic actions against the classical renin-angiotensin system axis in the cardiovascular system. However, its roles in the brain remain unclear. We examined possible roles of the angiotensin-converting enzyme 2/Ang-(1-7)/Mas axis in cognitive function, employing vascular cognitive impairment model mice. METHODS AND RESULTS: Male 10-week-old C57BL6 (wild-type mice, Mas1 knockout mice, Ang II type 2 receptor knockout mice, and Ang II type 2 receptor/Mas1 double knockout mice were subjected to bilateral carotid artery stenosis (BCAS) surgery. Six weeks after treatment, they were subjected to cognitive tasks. Brain samples were used for histopathological analysis. Cognitive function was significantly impaired in wild-type and double knockout mice after BCAS. On the other hand, the cognitive function of Mas1 knockout mice was maintained in spite of the reduction of cerebral blood flow with BCAS. Total cell number in the dentate gyrus region was significantly reduced after BCAS in wild-type but not in Mas1 knockout mice. The number of doublecortin-positive cells in the subgranular zone was not significantly different between wild-type and Mas1 knockout mice. Ang-(1-7) administration did not improve cognitive function in all mice after BCAS surgery. CONCLUSIONS: Lack of the Mas receptor may have a protective effect against chronic brain ischemia when the Ang II type 2 receptor exists.

Predicting outcome of Morris water maze test in vascular dementia mouse model with deep learning.

The Morris water maze test (MWM) is one of the most popular and established behavioral tests to evaluate rodents' spatial learning ability. The conventional training period is around 5 days, but there is no clear evidence or guidelines about the appropriate duration. In many cases, the final outcome of the MWM seems predicable from previous data and their trend. So, we assumed that if we can predict the final result with high accuracy, the experimental period could be shortened and the burden on testers reduced. An artificial neural network (ANN) is a useful modeling method for datasets that enables us to obtain an accurate mathematical model. Therefore, we constructed an ANN system to estimate the final outcome in MWM from the previously obtained 4 days of data in both normal mice and vascular dementia model mice. Ten-week-old male C57B1/6 mice (wild type, WT) were subjected to bilateral common carotid artery stenosis (WT-BCAS) or sham-operation (WT-sham). At 6 weeks after surgery, we evaluated their cognitive function with MWM. Mean escape latency was significantly longer in WT-BCAS than in WT-sham. All data were collected and used as training data and test data for the ANN system. We defined a multiple layer perceptron (MLP) as a prediction model using an open source framework for deep learning, Chainer. After a certain number of updates, we compared the predicted values and actual measured values with test data. A significant correlation coefficient was derived form the updated ANN model in both WT-sham and WT-BCAS. Next, we analyzed the predictive capability of human testers with the same datasets. There was no significant difference in the prediction accuracy between human testers and ANN models in both WT-sham and WT-BCAS. In conclusion, deep learning method with ANN could predict the final outcome in MWM from 4 days of data with high predictive accuracy in a vascular dementia model.

Roles of angiotensin II type 2 receptor in mice with fetal growth restriction.

Our previous report indicated that vascular injury enhances vascular remodeling in fetal growth restriction (FGR) mice. The angiotensin II type 2 receptor (AT2R) is relatively highly expressed in fetal mice. Therefore, we investigated the roles of AT2R in FGR-induced cardiovascular disease using AT2R knockout (AT2KO) mice. Dams (wild-type and AT2KO mice) were fed an isocaloric diet containing 20% protein (NP) or 8% protein (LP) until delivery. Arterial blood pressure, body weight, and histological changes in organs were investigated in offspring. The birth weight of offspring from dams fed an LP diet (LPO) was significantly lower than that of offspring from dams fed an NP diet. The heart/body and kidney/body weight ratios in AT2KO-LPO at 12 weeks of age were significantly higher than those in the other groups. Greater thickness of the left ventricular wall, larger cardiomyocyte size and enhancement of perivascular fibrosis were observed in AT2KO-LPO. Interestingly, mRNA expression of collagen I and inflammatory cytokines was markedly higher in the AT2KO-LPO heart at 6 weeks of age but not at 12 weeks of age. AT2R signaling may be involved in cardiovascular disorders of adult offspring with FGR. Regulation of AT2R could contribute to preventing future cardiovascular disease in FGR offspring.

Administration of bovine casein-derived peptide prevents cognitive decline in Alzheimer disease model mice.

There is a growing interest in identifying natural food ingredients that may serve to prevent dementia such as that due to Alzheimer disease (AD). Peptides derived from food proteins have been demonstrated to have various physiological activities such as a hypotensive action. Recent findings have indicated possible associations of hypertension with AD progression, and suggest that angiotensin converting enzyme (ACE) inhibitors with potential to pass through the blood brain barrier (BBB) may reduce the risk of AD. In this study, we investigated the effect of milk peptide (CH-3) on cognitive function in AD model mice. CH-3 contains a tripeptide (methionine-lysine-proline, MKP) that has been found to have a strong ACE inhibitory effect and the potential to pass through the BBB. Adult male ddY mice were used in this study, and an animal model of AD was induced by intracerebroventricular (ICV) injection of Aß1-42. CH-3 (250 mg/kg/day) or MKP (0.5 mg/kg/day) was orally administered every day starting 2 days before ICV injection. At 3 weeks after ICV injection, cognitive function was evaluated by the Morris water maze test. Brain samples were obtained after behavioral testing, and expression of inflammatory cytokines and NADPH oxidase subunits was measured by real-time quantitative RT-PCR. ICV injection of Aß1-42 significantly impaired cognitive function compared with that in PBS-injected mice. Daily administration of CH-3 markedly attenuated this Aß1-42-induced cognitive decline. Aß1-42 injection significantly enhanced the expression of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and p22phox in the mouse hippocampus compared with PBS injection, and showed a tendency to increase the expression of monocyte chemoattractant protein-1 (MCP-1), p47phox and gp91phox, whereas CH-3 treatment markedly reduced Aß1-42-induced TNF-α, MCP-1, iNOS, p47phox and gp91phox expression. Finally, administration of MKP also attenuated Aß1-42-induced cognitive impairment with an increase in cerebral blood flow. The present study demonstrated that repeated oral administration of CH-3 to AD model mice not only improved cognitive function but also suppressed the expression of inflammatory cytokines and production of oxidative stress, and suggests its therapeutic potential for preventing cognitive impairment in AD.

Enhancement of Adipocyte Browning by Angiotensin II Type 1 Receptor Blockade.

Browning of white adipose tissue (WAT) has been highlighted as a new possible therapeutic target for obesity, diabetes and lipid metabolic disorders, because WAT browning could increase energy expenditure and reduce adiposity. The new clusters of adipocytes that emerge with WAT browning have been named 'beige' or 'brite' adipocytes. Recent reports have indicated that the renin-angiotensin system (RAS) plays a role in various aspects of adipose tissue physiology and dysfunction. The biological effects of angiotensin II, a major component of RAS, are mediated by two receptor subtypes, angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R). However, the functional roles of angiotensin II receptor subtypes in WAT browning have not been defined. Therefore, we examined whether deletion of angiotensin II receptor subtypes (AT1aR and AT2R) may affect white-to-beige fat conversion in vivo. AT1a receptor knockout (AT1aKO) mice exhibited increased appearance of multilocular lipid droplets and upregulation of thermogenic gene expression in inguinal white adipose tissue (iWAT) compared to wild-type (WT) mice. AT2 receptor-deleted mice did not show miniaturization of lipid droplets or alteration of thermogenic gene expression levels in iWAT. An in vitro experiment using adipose tissue-derived stem cells showed that deletion of the AT1a receptor resulted in suppression of adipocyte differentiation, with reduction in expression of thermogenic genes. These results indicate that deletion of the AT1a receptor might have some effects on the process of browning of WAT and that blockade of the AT1 receptor could be a therapeutic target for the treatment of metabolic disorders.

Interferon regulatory factor 1 attenuates vascular remodeling; roles of angiotensin II type 2 receptor.

We previously reported interferon regulatory factor (IRF) 1 plays physiological roles in "growth"-regulated angiotensin II type 2 (AT2) receptor expression in fibroblasts. Here, we investigated whether IRF-1 is involved in attenuation of vascular remodeling in association with AT2 receptor upregulation. Neointimal area in injured artery after 14 days of cuff placement was significantly increased in IRF-1 knockout mice (IRF-1KO) and AT2 receptor knockout mice (AT2KO) compared with wild-type mice (WT: C57BL/6J). Treatment with compound 21 attenuated neointima formation in both WT and IRF-1KO. AT2 receptor mRNA expression after 7 days of cuff placement was significantly decreased in IRF-1KO compared with WT; however, IRF-1 expression did not differ between AT2KO and WT. Apoptotic changes in injured artery after 14 days of cuff placement were significantly attenuated in IRF-1KO, with a decrease in interleukin-1ß-converting enzyme and inducible nitric oxide synthase mRNA levels. These results indicate IRF-1 is one of the key transcriptional factors for the prevention of neointimal formation involving AT2 receptors.

Diabetic mice exhibited a peculiar alteration in body composition with exaggerated ectopic fat deposition after muscle injury due to anomalous cell differentiation.

BACKGROUND: Sarcopenic obesity, age-related muscle loss, which is compensated by an increase in fat mass, impairs quality of life in elderly people. Although the increase in intramuscular fat is associated with decreased insulin sensitivity and increased metabolic risk factors, the origin of diabetes-associated intramuscular fat has not been elucidated. Here, we investigated intramuscular fat deposition using a muscle injury model in type 2 diabetic mice. METHODS: Male 8-week-old C57BL/6 and 8-week-old and 26-week-old KKAy underwent intramuscular injection of cardiotoxin (Ctx) (100 µL/10 µM) into the tibialis anterior (TA) muscles. After 2 weeks, the muscles were removed and evaluated. RESULTS: KKAy exhibited impaired muscle regeneration and ectopic fat deposition. Such impairment was more marked in older KKAy. These changes were also observed in another diabetic mouse model, db/db and diet-induced obese mice but not in streptozocin-induced diabetic mice. Deposited fat was platelet-derived growth factor (PDGF) receptor alpha positive and its cytoskeleton was stained with Masson's trichrome, indicating it to be of fibro-adipocyte progenitor cell origin. Expression of a myogenic marker, myoD, was lower and that of PDGF receptor alpha and CCAAT/enhancer binding protein (CEBP) alpha was higher in Ctx-injured TA of KKAy compared with that of C57BL/6. Peroxisome proliferator-activated receptor γ (PPARγ) was highly expressed in fat-forming lesions in older KKAy. Treatment with all-trans retinoic acid prevented the formation of intramuscular fat; however, treatment with GW9662, a PPARγ antagonist, increased the fibrotic change in muscle. CONCLUSIONS: Diabetic mice showed impaired muscle regeneration with fat deposition, suggesting that diabetes may enhance sarcopenic obesity through a mechanism involving anomalous fibro-adipocyte progenitor cell differentiation.

Synergistic Inhibitory Effect of Rosuvastatin and Angiotensin II Type 2 Receptor Agonist on Vascular Remodeling.

We investigated the possibility that coadministration of rosuvastatin and compound 21 (C21), a selective angiotensin II type 2 (AT2) receptor agonist, could exert synergistic preventive effects on vascular injury. Vascular injury was induced by polyethylene cuff placement on the femoral artery in 9-week-old male C57BL/6J mice. Mice were treated with rosuvastatin and/or with C21 after cuff placement. Neointima formation was determined 14 days after the operation and cell proliferation, and superoxide anion production and expression of inflammatory cytokines were examined 7 days after cuff placement. Neointima formation was significantly attenuated by the treatment of rosuvastatin (5 mg kg(-1) day(-1)) or C21 (10 µg kg(-1) day(-1)), associated with the decreases in proliferating cell nuclear antigen (PCNA) labeling index, oxidative stress, and the expression of inflammatory markers. Treatment with a noneffective dose of rosuvastatin (0.5 mg kg(-1) day(-1)) plus a low dose of C21 (1 µg kg(-1) day(-1)) inhibited the PCNA labeling index, superoxide anion production, mRNA expressions of NAD(P)H subunits, and mRNA and protein expressions of inflammatory markers associated with marked inhibition of neointima formation. Angiotensin II type 1 (AT1) receptor mRNA expression did not differ the groups. By contrast, AT2 receptor mRNA expression was increased by administration of C21 at the dose of 10 µg kg(-1) day(-1) but not by C21 at the dose of 1 µg kg(-1) day(-1) or rosuvastatin. The combination of rosuvastatin and AT2 receptor agonist exerted synergistic preventive effects on vascular remodeling associated with the decreases in cell proliferation, oxidative stress, and inflammatory reaction. That could be a powerful approach to vascular disease prevention.