Atsushi Nagai

Transplantation of human mesenchymal stem cells promotes functional improvement and increased expression of neurotrophic factors in a rat focal cerebral ischemia model

Previous studies have suggested that intravenous transplantation of mesenchymal stem cells (MSCs) in rat ischemia models reduces ischemia‐induced brain damage. Here, we analyzed the expression of neurotrophic factors in transplanted human MSCs and host brain tissue in rat middle cerebral artery occlusion (MCAO) ischemia model. At 1 day after transient MCAO, 3 × 106 immortalized human MSC line (B10) cells or PBS was intravenously transplanted. Behavioral tests, infarction volume, and B10 cell migration were investigated at 1, 3, 7, and 14 days after MCAO. The expression of endogenous (rat origin) and exogenous (human origin) neurotorphic factors and cytokines was evaluated by quantitative real‐time RT‐PCR and Western blot analysis. Compared with PBS controls, rats receiving MSC transplantation showed improved functional recovery and reduced brain infarction volume at 7 and 14 days after MCAO. In MSC‐transplanted brain, among many neurotrofic factors, only human insulin‐like growth factor 1 (IGF‐1) was detected in the core and ischemic border zone at 3 days after MCAO, whereas host cells expressed markedly higher neurotrophic factors (rat origin) than control rats, especially vascular endothelial growth factor (VEGF) at 3 days and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) at 7 days after MCAO. Intravenously transplanted human MSCs induced functional improvement, reduced infarct volume, and neuroprotection in ischemic rats, possibly by providing IGF‐1 and inducing VEGF, EGF, and bFGF neurotrophic factors in host brain.

Microbleeds Are Associated With Subsequent Hemorrhagic and Ischemic Stroke in Healthy Elderly Individuals

Background and Purpose— Cerebral microbleeds (MBs) are frequently detected in patients with stroke, especially those who experience intracerebral hemorrhage. However, the clinical significance of MBs in subjects without cerebrovascular disease is still unclear. We performed a prospective study to determine whether the presence of MBs provides useful prognostic information in healthy elderly individuals. Methods— We tracked 2102 subjects (mean age, 62.1 years) over a mean interval of 3.6 years after they voluntarily participated in the brain checkup system at the Shimane Institute of Health Science. An initial assessment was performed to document the presence of MBs and silent ischemic brain lesions and to map the location of the MBs. During the follow-up period, we obtained information about stroke events that occurred in each subject. Results— MBs were detected in 93 of the 2102 subjects (4.4%). Strokes occurred in 44 subjects (2.1%) during the follow-up period. They were significantly more common among subjects with MBs. Age and hypertension were independent risk factors for MBs. The presence of MBs was more strongly associated with a deep brain hemorrhage (hazard ratio, 50.2; 95% CI, 16.7 to 150.9) than ischemic stroke (hazard ratio, 4.48; 95% CI, 2.20 to 12.2). All hemorrhagic strokes occurred in deep brain regions, and they were associated with MBs located in the deep brain region. Conclusions— This longitudinal study demonstrated that the presence of MBs can be used to predict hemorrhagic and ischemic stroke, even in healthy elderly individuals.

Metabolic Syndrome Is Associated With Silent Ischemic Brain Lesions

Background and Purpose— Metabolic syndrome (MetS) is a recognized risk factor for stroke, but it is unclear whether MetS is also related to subclinical ischemic lesions. We examined the association of MetS with the prevalence of silent brain infarction, periventricular hyperintensity, and subcortical white matter lesions in healthy adults. Methods— We conducted a cross-sectional study in 1151 Japanese healthy subjects. Three types of silent lesions were assessed by MRI scans. MetS was diagnosed using the criteria by the National Cholesterol Education Adult Treatment Panel III. Results— After adjusting for age and other factors, MetS was significantly associated with silent brain infarction, periventricular hyperintensity and subcortical white matter lesions. Among the MetS components, elevated blood pressure was commonly associated with all types of lesions. Dyslipidemia and elevated fasting glucose levels were associated with subcortical white matter lesions and periventricular hyperintensities, respectively. Positive trends were observed between the number of MetS components and prevalence of silent lesions. Conclusions— MetS is associated with the prevalence of silent lesions independent of other risk factors. The clustering of MetS components tends to increase the prevalence of silent lesions.

Upregulation of protease-activated receptor-1 in astrocytes in Parkinson disease: astrocyte-mediated neuroprotection through increased levels of glutathione peroxidase.

Abstract In the present study, we investigated the expression of protease-activated receptors (PARs), receptors for thrombin, in substantia nigra pars compacta (SNpc) of Parkinson disease (PD) brains and cultures of human neurons, astrocytes, oligodendrocytes, and microglia as determined by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of PAR-1 was demonstrated only in glial fibrillary acidic protein-positive astrocytes in SNpc, and the number of astrocytes expressing PAR-1 increased in SNpc of PD as compared with nonneurologic control brain. Immunoreactivity for thrombin and prothrombin was stronger in astrocytes and the vessel walls in SNpc of PD brains. PAR-1 was expressed in human astrocytes and neurons, but not in oligodendrocytes or microglia as determined by RT-PCR. We investigated thrombin-mediated activation of human astrocytes. Thrombin treatment activates human astrocytes and induces morphologic change and a marked increase in proliferation of astrocytes. Increased expression of glial cell line-derived growth factor and glutathione peroxidase (GPx) but no change in the expression of nerve growth factor and inflammatory cytokines/chemokine (IL-1β, IL-6, IL-8, MCP-1) was found in thrombin/PAR-activated astrocytes. Next, we studied the neuroprotective effect exerted by thrombin-activated astrocytes in human cerebral neuron × human neuroblastoma hybrid neurons. Although thrombin showed neurotoxicity against human hybrid neurons in a dose-dependent manner, the conditioned media derived from thrombin-pretreated astrocyte cultures promoted the survival of human hybrid neurons. The protective effect was completely inhibited with a GPx inhibitor, mercaptosuccinic acid, indicating that GPx released from thrombin/PAR-activated astrocytes is responsible for neuroprotection of hybrid neurons against thrombin cytotoxicity. The present study suggests that the increased expression of PAR-1 in astrocytes in SNpc of PD brain is the restorative move taken by the brain to provide neuroprotection against neuronal degeneration and cell death of dopaminergic neurons caused by noxious insults during the progression of PD pathology.

Human Microglia Transplanted in Rat Focal Ischemia Brain Induce Neuroprotection and Behavioral Improvement

Background and Purpose Microglia are resident immunocompenent and phagocytic cells of central nervous system (CNS), which produce various cytokines and growth factors in response to injury and thereby regulate disease pathology. The purpose of this study is to investigate the effects of microglial transplantation on focal cerebral ischemia model in rat. Methods Transient middle cerebral artery occlusion (MCAO) in rats was induced by the intraluminal filament technique. HMO6 cells, human microglial cell line, were transplanted intravenously at 48 hours after MCAO. Functional tests were performed and the infarct volume was measured at 7 and 14 days after MCAO. Migration and cell survival of transplanted microglial cells and host glial reaction in the brain were studied by immunohistochemistry. Gene expression of neurotrophic factors, cytokines and chemokines in transplanted cells and host rat glial cells was determined by laser capture microdissection (LCM) and quantitative real time-PCR. Results HMO6 human microglial cells transplantion group demonstrated significant functional recovery compared with control group. At 7 and 14 days after MCAO, infarct volume was significantly reduced in the HMO group. In the HMO6 group, number of apoptotic cells was time-dependently reduced in the infarct core and penumbra. In addition, number of host rat microglia/macrophages and reactive astrocytes was significantly decreased at 7 and 14 days after MCAO in the penumbra. Gene expression of various neurotrophic factors (GDNF, BDNF, VEGF and BMP7) and anti-inflammatory cytokines (IL4 and IL5) was up-regulated in transplanted HMO6 cells of brain tissue compared with those in culture. The expression of GDNF and VEGF in astrocytes in penumbra was significantly up-regulated in the HMO6 group. Conclusions Our results indicate that transplantation of HMO6 human microglial cells reduces ischemic deficits and apoptotic events in stroke animals. The results were mediated by modulation of gliosis and neuroinflammation, and neuroprotection provided by neurotrophic factors of endogenous and transplanted cells-origin.

Mesenchymal stem cell transplantation modulates neuroinflammation in focal cerebral ischemia: Contribution of fractalkine and IL-5

Mesenchymal stem cells (MSCs) are reported to possess immunomodulatory properties. Previous reports have demonstrated the beneficial effects of MSC-transplantation in focal cerebral ischemia animal models. In this study, we have investigated the neuroimmunomodulatory functions of human MSCs, transplanted in a rat focal ischemia model of transient middle cerebral artery occlusion (MCAO). Our results revealed that in a human mesenchymal stem cell line, B10 cell transplantation decreased the accumulation of Iba-1(+) microglia and GFAP(+) astrocytes, and inhibited proinflammatory gene expression in the core and ischemic border zone (IBZ). Among the proinflammatory genes iNOS, which was expressed in microglia/macrophage, was persistently inhibited up to 7days after MCAO. In vivo laser capture microdissection and double immunofluorescence staining, and in vitro B10 cell culture experiments showed that, in inflammatory conditions, B10 cells expressed cytokines and growth factors including IL-5, fractalkine, IGF-1, GDNF and VEGF. Fractalkine and IL-5 inhibited cytokine-induced proinflammatory gene expression including iNOS in a human microglia cell line. Thus, our results demonstrate that MSC transplantation suppresses MCAO focal ischemia-induced inflammation, possibly through expression of fractalkine and IL-5.

Age-Related Changes in White Matter Lesions, Hippocampal Atrophy, and Cerebral Microbleeds in Healthy Subjects Without Major Cerebrovascular Risk Factors

Although cumulative evidence indicates that risk factors for arteriosclerosis have an impact on age-related changes in brain pathology, the influence of aging without major risk factors on changes in brain structures has not yet been fully elucidated. We used magnetic resonance imaging (MRI) to study how aging affects structural changes in the brain (eg, white matter lesions, hippocampal atrophy [HA], microbleeds) in normal subjects without major risk factors for cerebrovascular diseases. We studied 1108 subjects who underwent voluntary brain screening and had no cerebrovascular risk factors, such as hypertension, diabetes mellitus, or hyperlipidemia. We examined the conventional and T2-weighted MRI to define white matter hyperintensities, HA, and cerebral microbleeds in addition to all physical parameters, blood biochemical data, and neuropsychiatric symptoms. We found that the prevalence of white matter lesions and HA increased significantly with age (P < .001). Logistic analysis showed that periventricular hyperintensity was significantly related to age (P < .0001) and depressive state (P < .01). A linear relation was found between white matter lesions and HA (P < .05). Cerebral microbleeds also increased with age, and their presence was associated with HA (P < .001). White matter lesions, HA, and cortical microbleeds were associated with one another in healthy elderly subjects, and these changes were affected by the aging process independent of any cerebrovascular risk factors. Cerebral amyloid angiopathy may underlie these age-related brain changes.

Cathepsin B and H activities and cystatin C concentrations in cerebrospinal fluid from patients with leptomeningeal metastasis

BACKGROUND Cysteine proteases are involved in the extension of cancer into the subarachnoid space. The presence of cathepsins B and H along with their potent inhibitor cystatin C in the cerebrospinal fluid (CSF) was investigated in patients with leptomeningeal metastasis of cancer (LM). MATERIALS AND METHODS CSF samples were obtained in 16 cases of LM (10 solid tumors and 6 leukemia or lymphoma) and compared with 11 cancer cases without involvement of the central nervous system, 12 multiple sclerosis cases and 34 healthy volunteers. The activity of the enzymes was measured, their molecular forms were analyzed by the Western blotting, and the concentration of cystatin C was measured by ELISA. Immunohistochemistry of the leptomeningeal tissues was also performed in six autopsy cases of LM. RESULTS High activities of cathepsins B and H along with decreased cystatin C concentration were observed in CSF of LM as compared with three control groups. Western blot analysis revealed higher concentration of the enzyme protein as well as its active forms in samples with higher enzyme activity. Cells metastasizing leptomeningeal tissue were clearly positive in immunohistochemical staining of cathepsins, indicating active production by tumor cells. CONCLUSION Production of cathepsins B and H by tumor cells and their high activity along with concomitant decrease of their potent inhibitor, cystatin C, in the CSF might contribute in the process of metastasis and spread of the cancer cells in the leptomeningeal tissues. A high enzyme activity/cystatin C concentration ratio in the CSF could be useful when diagnosing LM in combination with other parameters.

Association of cognitive dysfunction with hippocampal atrophy in elderly Japanese people with type 2 diabetes

This study aimed to examine the associations of cognitive function with hippocampal and whole brain atrophies, age, education, and diabetes-related parameters in patients with type 2 diabetes. Sixty-one patients over 65 years of age with type 2 diabetes and 53 age- and sex-matched non-diabetic controls were enrolled. Hippocampal and whole brain atrophies were assessed by quantifying hippocampal and brain volumes by brain magnetic resonance imaging. Cognitive function was evaluated by Mini-Mental State Examination (MMSE) and the Revised Hasegawa Dementia Scale (HDS-R). Compared with the non-diabetic group, patients with type 2 diabetes showed significant increases in hippocampal and whole brain atrophies. The MMSE and HDS-R scores in type 2 diabetic patients showed significant negative correlations with age and significant positive correlations with years of education. These scores were also significantly negatively correlated with hippocampal atrophy, but not whole brain atrophy. Hippocampal atrophy in diabetic patients did not, however, correlate with age, years of education, or diabetes-related parameters. We showed hippocampal and whole brain atrophies to be more frequent in elderly patients with type 2 diabetes than in non-diabetic controls. Their cognitive functions were significantly and negatively associated with hippocampal atrophy.

Effective ex vivo expansion of hematopoietic stem cells using osteoblast‐differentiated mesenchymal stem cells is CXCL12 dependent

Effective ex vivo expansion of hematopoietic stem cells (HSCs) is a prerequisite for HSC transplantation. Growth and maintenance of HSC is dependent on cytokine and niche factors. We investigated whether mesenchymal stem cells (MSCs) or osteogenic cytokine‐differentiated MSCs play a role in HSC expansion. We used the human HM3.B10 (B10) MSC cell line and the osteoblast‐differentiated B10 (Ost‐B10) as a feeder layer and examined ex vivo expansion of CD34+CD38− HSCs obtained from peripheral blood (PB) and cord blood (CB) with or without several growth cytokines. Both undifferentiated B10 and Ost‐B10 cells exhibited similar effects on total HSC expansion; however, Ost‐B10 demonstrated a higher potency in CD34+CD38− cell‐specific proliferation in the presence of cytokines compared to undifferentiated B10 HSCs. Colony‐forming cell assay and long‐term culture initiating cell assay revealed that Ost‐B10 displayed multipotent differentiation and enabled long‐term ex vivo culture of HSCs. We next examined the relationship between HSC expansion and the presence of various chemokines. CXCL4 and CXCL12 expression were increased in Ost‐B10 cells compared with the B10 cells. CD34+CD38− cells were significantly increased with CXCL12, but not CXCL4 treatment. siRNA inhibition of CXCL12 decreased CXCL12 secretion in both B10 and Ost‐B10, whereas expansion of CD34+CD38− cells was decreased in Ost‐B10 alone. These results demonstrated that ex vivo expansion of HSCs may be highly effective through osteoblast‐differentiated MSCs acting as a feeder layer, and likely operates through the CXCL12 chemokines signaling pathway.