Serum microRNA-501-3p is a potential diagnostic tool for detecting mild cognitive impairment: Ehime genome study.

Tight junction disruption and dysfunction are involved in the progression of blood-brain barrier (BBB) breakdown. Recent investigations have revealed BBB disruption in patients with vascular cognitive decline. Our previous studies showed that miR-501-3p negatively regulates cerebral endothelial tight junction protein-1, resulting in the disruption of the BBB, and playing an important role in the development of vascular cognitive impairment. BBB breakdown in white matter lesions is often seen in the patients with vascular mild cognitive impairment (MCI). We therefore hypothesize that most early-phase MCI patients may demonstrate elevated expression of miR-501-3p and sought to investigate whether serum exosome miR-501-3p levels could be a clinical indicator for detecting mild cognitive impairment. One hundred and seventy-eight subjects (aged 73 [68-75] years, 53% male) were recruited for this study. The Japanese version of the Montreal Cognitive Assessment (MoCA-J) was used for detecting MCI. Serum exosome miR-501-3p expression levels were measured by qPCR methods. Patients were divided into two groups depending on whether their miR-501-3p ∆Ct values were above ("High"; n = 74) or below ("Low"; n = 104) cutoff levels determined by ROC curve. MCI was detected significantly more often in the miR-501-3p-High group (vs. -Low group, 63.5% vs. 47.1%, respectively; p < 0.05). Multivariate logistic regression analysis showed a significant association between MCI status and High miR-501-3p (odds ratio 2.662; p < 0.01), improved vs. known risk factors. In non-diabetic patients, High miR-501-3p was positively associated with MCI status (odds ratio 3.633; p < 0.01) and also positively associated with MCI status in those with atherosclerosis (odds ratio 3.219; p < 0.01). The present study demonstrates that elevated expression of blood exosomal miR-501-3p can indicate the presence of MCI in human patients. Early detection of vascular injuries may allow a reduction in progressive dementia through the management of vascular risk factors.

Generation of DNA-aptamers targeting galectin-7 for the identification of cholesteatoma residue.

PURPOSE: Aiming at complete excision of cholesteatoma during trympanomastoidectomy and therefore reducing the risk of recurrence, intraoperative imaging techniques are required to assist the visualization of cholesteatoma residue. Galectin-7 has been demonstrated to be a biomarker for cholesteatoma matrix and used for intraoperatively identifying the excision margins. METHODS: A galectin-7-targeted DNA-aptamer library was generated for labeling the cholesteatoma matrix using cell-systematic evolution of ligands by an exponential enrichment technique. The binding characteristics of the identified aptamers were analyzed, and structure optimization of the identified aptamers was carried out both in silico and in vitro. FINDINGS: A fluorophore-labeled structure-optimized DNA fragment was commercially synthesized as a non-invasive aptamer-based probe for intraoperative lesion detection. Using galectin-7-aptamer-guided molecular imaging, the excision margins of cholesteatoma matrix and surrounding normal tissue were successfully achieved within 15-20 min. CONCLUSIONS: Galectin-7-targeted aptamers could benefit molecular imaging-guided surgical treatment, which would enable clinicians to not only intraoperatively detect the locations of cholesteatoma matrix in the middle ear, but also assess the postoperative response of the expression profile to therapy. It is highly expected that further efforts for rational design and development should be directed towards the development of clinically translatable aptamer-based imaging agents.

Inhibition of Histamine Release from RBL-2H3 Cells by Zoledronate Did Not Affect Rab27a/Doc2a Interaction.

Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca2+) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca2+ concentration and Ca2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca2+ influx.

Toxicokinetics of methylmercury in diabetic KK-Ay mice and C57BL/6 mice.

We compared the toxicokinetics of methylmercury (MeHg) in KK-Ay type 2 diabetic mice and C57BL/6J mice to evaluate how metabolic changes associated with diabetes affect MeHg toxicokinetics. A single dose of MeHg (0.2, 1, or 5 mg mercury/kg) was administered orally to 12-week-old KK-Ay and C57BL/6J male mice. Total mercury concentrations in plasma, blood cells, whole blood, and tissues (brain, kidneys, liver, and pancreas) were measured after 4, 7, 11, and 14 days. The volume of distribution/bioavailability and the elimination rate constant per day were higher in KK-Ay mice, while the terminal elimination half-life was lower in almost all samples of KK-Ay mice. The area under the curve was lower in all blood and almost all tissue samples from KK-Ay mice. Total clearance/bioavailability was lower in all blood and tissue samples of KK-Ay mice at all MeHg doses. These results indicate that MeHg is more rapidly absorbed by, and eliminated from, the blood cells, brain, liver, kidney, and pancreas of KK-Ay mice under the experimental conditions. Different patterns of tissue-to-plasma and tissue-to-whole blood partition coefficients suggest that notable differences in MeHg transfer between plasma and blood cells affect its distribution in tissues of the two mouse strains. These findings are useful to understand the selective distribution of MeHg to target organs and the sensitivity to MeHg in pathological states.

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.

Tachykinin-1 receptor antagonism suppresses substance-P- and compound 48/80-induced mast cell activation from rat mast cells expressing functional mas-related GPCR B3.

OBJECTIVE: Mice and rats are important animal models for mast cell (MC) study. However, rat Mas-related-GPCR-B3 receptor (MRGPRB3) has been less studied than its mouse counterpart. Therefore, we aimed to characterize rat MRGPRB3. METHODS: Mrgprb3 mRNA expression was assessed in peritoneal cells (RPCs) and peritoneal MCs (RPMCs) of wild-type rats, RPCs of MC-deficient rats, and RBL-2H3 cells by reverse-transcriptase polymerase chain reaction (RT-PCR). RPMCs, MRGPRX2-transfected and non-transfected RBL-2H3 cells were activated by 15-30 min incubation with DNP-BSA, substance-P (SP), or compound-48/80. L732138 or CP96344 was used as a tachykinin/neurokinin-1-receptor antagonist. Histamine release from MCs was measured by HPLC fluorometry. RESULTS: Mrgprb3 mRNA expression was found in all cells, with the highest level in wild-type RPCs. All cells responded to DNP-BSA, but only MRGPRX2-transfected-RBL-2H3 cells and RPMCs responded to all activators. L732138 (0.1-10 µM) and CP96344 (1-100 µM) suppressed SP (10 µM)-induced RPMC activation. L732138 inhibition was dose independent, whereas CP96344 inhibition occurred in a dose-dependent manner. Additionally, only CP96344 suppressed SP (100 µM)- and compound-48/80 (10 µg/mL)-induced RPMC activation. CONCLUSIONS: RPMCs expressing functional MRGPRB3 response upon MRGPRX2 ligands to regulated MC-mediated activities. It`s provide novel insights for future pseudo-allergic studies in rodents.

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.

Angiotensin 1-7 alleviates aging-associated muscle weakness and bone loss, but is not associated with accelerated aging in ACE2-knockout mice.

The angiotensin-converting enzyme 2 (ACE2)-angiotensin 1-7 (A1-7)-A1-7 receptor (Mas) axis plays a protective role in the renin-angiotensin system (RAS). We recently found that ACE2 knockout (ACE2KO) mice exhibit earlier aging-associated muscle weakness, and that A1-7 alleviates muscle weakness in aging mice. In the present study, we investigated the role of the A1-7-Mas pathway in the effect of ACE2 on physiological aging. Male wild-type, ACE2KO, and Mas knockout (MasKO) mice were subjected to periodical grip strength measurement, followed by administration of A1-7 or vehicle for 4 weeks at 24 months of age. ACE2KO mice exhibited decreased grip strength after 6 months of age, while grip strength of MasKO mice was similar to that of wild-type mice. A1-7 improved grip strength in ACE2KO and wild-type mice, but not in MasKO mice. Muscle fibre size was smaller in ACE2KO mice than that in wild-type and MasKO mice, and increased with A1-7 in ACE2KO and WT mice, but not in MasKO mice. Centrally nucleated fibres (CNFs) and expression of the senescence-associated gene p16INK4a in skeletal muscles were enhanced only in ACE2KO mice and were not altered by A1-7. ACE2KO mice, but not MasKO mice, exhibited thinning of peripheral fat along with increased adipose expression of p16INK4a A1-7 significantly increased bone volume in wild-type and ACE2KO mice, but not in MasKO mice. Our findings suggest that the impact of ACE2 on physiological aging does not depend on the endogenous production of A1-7 by ACE2, while overactivation of the A1-7-Mas pathway could alleviate sarcopenia and osteoporosis in aged mice.

Therapeutic perspective on vascular cognitive impairment.

Dementia is one of the greatest public health concerns for the modern aging world. Over the last decade, most researchers developing new therapeutic strategies for dementia have focused on amyloid-ß. In contrast, numerous recent studies have indicated that vascular risk factors are associated with various forms of dementia, and that in fact most forms of dementia can be considered an extension of vascular disease. Accordingly, it is sensible to pursue treatment approaches that focus on the blood vessels. Blood-brain barrier (BBB) disruptions in the white matter of patients with vascular cognitive impairment (VCI) have been observed using imaging analysis, and might be potential targets for novel VCI treatment. Tight junctions between cerebral endothelial cells play an important role in the function of the BBB, and recent studies have demonstrated the essential role of microRNAs in regulating tight junctions. Further elucidation of the mechanisms of tight junction-disruption in dementia are likely to lead to promising novel treatments. In this article, we summarize current knowledge regarding microRNAs and vascular cognitive impairment and the possibility of utilizing microRNAs as biomarkers for BBB dysfunction, and seek to envision future therapeutic strategies.

Controlled isoflurane anesthesia exposure is required for reliable behavioral testing in murine surgical models.

We investigated the effects of variations in anesthesia exposure time prior to conducting anxiety response behavioral testing in sham controls from an experimental murine model. The staying time in the center area of the Open Field test in the "long exposure" group was significantly decreased compared to that of the "short exposure" group. Significant correlation was found between anesthesia time and the duration of staying time in the center area. We conclude that anesthesia time may have a significant impact on behavioral anxiety testing in this context, and advise careful control of this parameter in protocol optimization in related surgical animal models.

Evaluation of neurobehavioral impairment in methylmercury-treated KK-Ay mice by dynamic weight-bearing test.

Methylmercury (MeHg) is known to cause neurobehavioral impairment in human and experimental animals. We previously reported that MeHg (5 mg Hg/kg) induced severe neurobehavioral dysfunction in 4-week-old KK-Ay mice, although it is difficult to evaluate quantitatively the neurobehavioral impairment in MeHg-treated KK-Ay mice because of their obesity. The aim of this study was to evaluate MeHg-induced neurobehavioral dysfunction in KK-Ay mice using the dynamic weight-bearing test, which analyzes the animal's weight distribution between the four limbs. Male 12-week-old KK-Ay mice were treated with MeHg (5 mg Hg/kg) three times per week for 5 weeks. Body weight loss began after approximately 2 weeks of MeHg treatment, and decreased significantly at 4 weeks. Seven of the nine MeHg-treated mice exhibited overt neurological symptoms such as ataxia and gait disturbance. The weight-bearing load was lower for the forelimb than for the hindlimb at baseline and until 1 week after MeHg treatment was initiated. In weeks 2-4, the dynamic weight-bearing loads on the forelimb and hindlimb were similar. The load on the forelimb exceeded the load on the hindlimb after 5 weeks of treatment. This finding indicates that the dynamic weight-bearing test is useful for semi-quantitative evaluation of neurobehavioral impairment in MeHg-treated rodents, and is less stressful for the animals. Infiltration of CD204-positive macrophages was observed in the sciatic nerve of MeHg-treated mice, suggesting that CD204 can serve as a useful marker of tissue injury in peripheral nerves and a possible target in regenerating peripheral nerves and controlling neuropathies.

[Milk, Daily products and Bone health.Effect of Milk or Milk Protein on Cognitive Function.]

Increase in milk and dairy intake reduces the development of all-cause dementia, especially Alzheimer's disease(AD)in elderly Japanese population from the Hisayama study. Recent meta-analysis also indicates that beneficial effect of milk and dairy intake on cognitive disorders is significantly observed in Asian population. We have reported that oral administration of milk peptide, CH-3 to AD model mice not only improved cognitive function but also suppressed the expression of inflammatory cytokines and production of oxidative stress. Moreover, administration of other milk proteins improves cognitive function or outcome in AD patients. These findings indicate that milk and dairy intake have possible anti-dementia effects. Further analysis should be necessary to overcome the explosion of dementia worldwide with lifestyle modification.

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.

Role of the renal sympathetic nerve in renal glucose metabolism during the development of type 2 diabetes in rats.

AIMS/HYPOTHESIS: Recent clinical studies have shown that renal sympathetic denervation (RDX) improves glucose metabolism in patients with resistant hypertension. We aimed to elucidate the potential contribution of the renal sympathetic nervous system to glucose metabolism during the development of type 2 diabetes. METHODS: Uninephrectomised diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats underwent RDX at 25 weeks of age and were followed up to 46 weeks of age. RESULTS: RDX decreased plasma and renal tissue noradrenaline (norepinephrine) levels and BP. RDX also improved glucose metabolism and insulin sensitivity, which was associated with increased in vivo glucose uptake by peripheral tissues. Furthermore, RDX suppressed overexpression of sodium-glucose cotransporter 2 (Sglt2 [also known as Slc5a2]) in renal tissues, which was followed by an augmentation of glycosuria in type 2 diabetic OLETF rats. Similar improvements in glucose metabolism after RDX were observed in young OLETF rats at the prediabetic stage (21 weeks of age) without changing BP. CONCLUSIONS/INTERPRETATION: Here, we propose the new concept of a connection between renal glucose metabolism and the renal sympathetic nervous system during the development of type 2 diabetes. Our data demonstrate that RDX exerts beneficial effects on glucose metabolism by an increase in tissue glucose uptake and glycosuria induced by Sglt2 suppression. These data have provided a new insight not only into the treatment of hypertensive type 2 diabetic patients, but also the pathophysiology of insulin resistance manifested by sympathetic hyperactivity.

Platelet-endothelial cell interaction in brain microvessels of angiotensin II type-2 receptor knockout mice following transient bilateral common carotid artery occlusion.

The purpose of this study was to investigate the behavior of platelets (rolling and adhesion) in cerebral microvessels of angiotensin II type-2 receptor-knockout (AT2RKO) mice after transient bilateral carotid artery occlusion using intravital fluorescence microscopy. Twenty AT2RKO mice, consisting of 11 mice in the sham group and 9 mice in the ischemia reperfusion group (reperfusion after 15 min of bilateral, total carotid artery occlusion) were used in this study. The hole traversed the bone and dura mater, but arachnoid, pia mater, and cerebral parenchyma were preserved. Platelets were harvested from donor mice and stained using carboxyfluorescein diacetate succinimidyl ester. The number of platelets showing rolling and adhesion to pial vessels in AT2 deficient mice at 3 and 6 h after cerebral ischemia reperfusion was significantly higher than that in the sham group (P < 0.05). In addition, AT2 receptor has an inhibitory role in platelet rolling and adhesion after cerebral ischemia reperfusion.

Institutional Review Board Considerations for Clinical Trials.

To protect subjects who participate in human research, Institutional Review Boards (IRBs) play an important role in reviewing research and determining the validity of a study by comprehensively examining it for ethical issues, including invasiveness and management of personal information. They conduct regular and independent reviews to protect the health, rights, and welfare of research subjects. When we as researchers conduct clinical research, we must obtain IRB approval and submit our research for investigation of ethical issues before we begin.

Animal Models of Vasculitis.

The diagnosis of vasculitis in rheumatoid arthritis (RV) is associated with considerable mortality; therefore, understanding the basic mechanisms underlying the pathogenesis of vasculitis is very important. Animal models of vasculitis have contributed to elucidating such mechanisms. We here introduce a Candida albicans water-soluble glycoprotein (CAWS)-induced vasculitis model and the methodological approach to evaluate inflammatory vascular change.