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Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling., Graphical Abstract, Yahata et al. refined a previous G13513A-mpTALEN to more efficiently manipulate mtDNA heteroplasmy in MELAS-iPSCs. Mutation-rich MELAS-iPSCs showed unusual differentiation properties, which were recovered by decreasing the heteroplasmy levels with new G13513A-mpTALEN. They further demonstrated that drug-inducible, MYOD-transfected MELAS-iPSCs could efficiently differentiate into MyHC-positive myocytes, with or without mutant mtDNA.

Induced pluripotent stem cells (iPSCs) are suitable for studying mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations. Here, we generated iPSCs from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with the m.13513G>A mutation. The patient’s dermal fibroblasts were reprogrammed, and we established two iPSC clones with and without mutant mtDNA. Furthermore, we tried to decrease mutant mtDNA level in iPSCs using transcription activator-like effector nucleases (TALENs). We originally engineered platinum TALENs, which were transported into mitochondria, recognized the mtDNA sequence including the m.13513 position, and preferentially cleaved G13513A mutant mtDNA (G13513A-mpTALEN). The m.13513G>A heteroplasmy level in MELAS-iPSCs was decreased in the short term by transduction of G13513A-mpTALEN. Our data demonstrate that this mtDNA-targeted nuclease would be a powerful tool for changing the heteroplasmy level in heteroplasmic iPSCs, which could contribute to elucidation of the pathological mechanisms of mitochondrial diseases caused by mtDNA mutations.

As a proof of concept that removal of blood amyloid-β (Aβ) can reduce Aβ deposition in the brains of patients with Alzheimer's disease, cortices of patients who had undergone hemodialysis (HD), which removes Aβ from the blood, were histochemically analyzed; postmortem brain sections were stained with anti-Aβ antibodies. Brains from patients who had undergone HD had significantly fewer senile plaques than those of patient who had not undergone HD. This significant difference was also confirmed by silver staining. Our findings suggest that removal of blood Aβ by hemodialysis results in lower accumulation of Aβ in the brain.

Oligomeric forms of amyloid-β peptide (Aβ) are thought to play a pivotal role in the pathogenesis of Alzheimer's disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. Aβ oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693Δ mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated Aβ oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs., Cell Stem Cell, 12(4), pp.487-496; 2013

The organic cation transporters OCT1, 2, and 3 (SLC22A1-3) have been implicated in the elimination of biogenic amines such as histamine. Among them, OCT3 was identified as an uptake-2 transporter, responsible for clearance of histamine. Because increasing evidence suggests the involvement of histamine in cerebral ischemia, we investigated the effects of targeted disruption of organic cation transporter-3 (Oct3) on the severity of ischemic brain damage. Transient focal ischemia for 1 hour was induced by occlusion of the middle cerebral artery (MCA) of homozygous Oct3-deficient mice and their wild-type (Wt) littermates. Although targeted disruption of Oct3 did not affect physiological parameters after MCA occlusion, this disruption significantly increased histamine content in the ischemic cortex and significantly reduced the infarct volume after cerebral ischemia. Furthermore, targeted disruption of Oct3 prevented the reduction of regulatory T-cell proportion after cerebral ischemia while this disruption did not affect Th1 and Th2 cells proportions after ischemia. Since repeated administration of L-histidine (a precursor of histamine) to Wt mice also showed the same effects, our observations suggested that OCT3 is the molecule responsible for clearance of ischemia-induced histamine in the brain and targeted disruption of Oct3 ameliorated ischemic brain damage through an increase in regulatory T cells.

Circulating bone marrow-derived immature cells, including endothelial progenitor cells, have been implicated in homeostasis of the microvasculature. Decreased levels of circulating endothelial progenitor cells, associated with aging and/or cardiovascular risk factors, correlate with poor clinical outcomes in a range of cardiovascular diseases. Herein, we transplanted bone marrow cells from young stroke-prone spontaneously hypertensive rats (SHR-SP) into aged SHR-SP, the latter not exposed to radiation or chemotherapy. Analysis of recipient peripheral blood 28 days after transplantation revealed that 5% of circulating blood cells were of donor origin. Cerebral infarction was induced on day 30 posttransplantation. Animals transplanted with bone marrow from young SHR-SP displayed an increase in density of the microvasculature in the periinfarction zone, reduced ischemic brain damage and improved neurologic function. In vitro analysis revealed enhanced activation of endothelial nitric oxide synthase and reduced activation p38 microtubule-associated protein (MAP) kinase, the latter associated with endothelial apoptosis, in cultures exposed to bone marrow-derived mononuclear cells from young animals versus cells from aged counterparts. Our findings indicate that partial rejuvenation of bone marrow from aged rats with cells from young animals enhances the response to ischemic injury, potentially at the level of endothelial/vascular activation, providing insight into a novel approach ameliorate chronic vascular diseases.

Induced pluripotent stem cells (iPSCs) are suitable for studying mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations. Here, we generated iPSCs from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with the m.13513GA mutation. The patient's dermal fibroblasts were reprogrammed, and we established two iPSC clones with and without mutant mtDNA. Furthermore, we tried to decrease mutant mtDNA level in iPSCs using transcription activator-like effector nucleases (TALENs). We originally engineered platinum TALENs, which were transported into mitochondria, recognized the mtDNA sequence including the m.13513 position, and preferentially cleaved G13513A mutant mtDNA (G13513A-mpTALEN). The m.13513GA heteroplasmy level in MELAS-iPSCs was decreased in the short term by transduction of G13513A-mpTALEN. Our data demonstrate that this mtDNA-targeted nuclease would be a powerful tool for changing the heteroplasmy level in heteroplasmic iPSCs, which could contribute to elucidation of the pathological mechanisms of mitochondrial diseases caused by mtDNA mutations.

Syntaxin1 and synaptotagmin are located in the pre-synaptic terminals and play central roles in Ca2+-triggered neurotransmitter release. Because excessive synaptic transmission has been implicated in neuronal cell death after ischemia, we investigated the effects of cerebral ischemia on the levels of these proteins using a rat permanent focal ischemia model. Western blot analysis revealed that the protein level of syntaxin1 was significantly up-regulated in the ischemic core cortex and peri-ischemic cortex at 1 day after ischemia, while the protein level of synaptotagmin was not. Immunohistochemical analysis revealed that the protein level of syntaxin1 was markedly up-regulated in the ischemic areas where immunoreaction for MAP2 was lost. Furthermore, we showed that resident microglial cells were quite vulnerable to ischemia. Our data provide novel insights into the molecular mechanism of cerebral ischemia at the pre-synaptic terminals.

The effect of postischemic mild hypothermia on the inner ear has not been clarified. In this study, we investigated whether hypothermia after transient ischemia could prevent cochlear damage and its therapeutic time window. Mongolian gerbils were divided into six groups: a sham-operation group, a normothermia group, and four hypothermia groups in which hypothermia was induced 1-7, 1-4, 3-6, and 6-9 h after reperfusion. Animals subjected to postischemic mild hypothermia within 3 h after reperfusion had attenuated hearing loss and inner hair cell loss. The protective effect was greater when hypothermia was induced earlier and had a longer duration. This implies that mild hypothermia after ischemia could have therapeutic effects for inner ear ischemic damage.

The effects of transient cochlear ischemia on spiral ganglion cells (SGCs) were studied in Mongolian gerbils. Ischemic insult was induced by occluding the bilateral vertebral arteries of gerbils for 15 min. Seven days after ischemia, the percentage of SGCs decreased to 67.5% from the preischemic baseline in the basal turn. Evaluation with immunohistochemical staining showed TUNEL-positive reactions in the SGCs with fragmented nuclei. In addition, we investigated the protective effects of ginsenoside Rb1 (gRb1) against ischemic injury to SGCs. Seven days after ischemia, the auditory brainstem response threshold shift was significantly reduced and the percentage of SGCs decreased to 90.2% from the preischemic baseline in the basal turn in the gRb1-treated group. These findings suggest that gRb1 prevented hearing loss caused by ischemic injury to SGCs in Mongolian gerbils.

Background: Red ginseng root (Panax Ginseng C.A. Meyer) has been used clinically by many Asian people for thousands of years without any detrimental effects. One of the major components of red ginseng root is ginsenoside Rb1 (gRb1, C54H92O23, molecular weight 1109.46). Previously, we showed that intravenous infusion of gRb1 attenuated ischemic brain damage in rats through up-regulation of an anti-apoptotic factor, Bcl-xL. In the present study, we have investigated whether intravenous infusion of gRb1attenuates spinal cord injury or not. Methods: Adult male Wistar rats aged 12-14 weeks, weighing 250-300g, were used. Spinal cord injury (SCI) was induced by compressing the thoracic cord (Th12) with a 20 g weight for 20 minutes. Thirty minutes later, a total of 60µl gRb1 solution (12 or 60µg/60µl in saline) or the same volume of vehicle (saline) was infused once into the left femoral vein. Subsequently, continuous intravenous administration of gRb1 was performed for 7 days. The locomotor function was assessed using the Basso-BeattieBresnahan (BBB) score and the open field test at 1, 3 or 7days after SCI. The animals were killed at 7 days after SCI and the SCI lesions were assessed histologically. Results: Post-traumatic intravenous infusion of gRb1 (12µg/day or 60µg/day) ameliorated BBB score, rearing activity and neuronal density in the anterior horn of the damaged spinal cord at 7 days after SCI. Subsequent studies that focused on gRb1-induced expression of gene products responsible for neuronal death or survival revealed that gRb1 upregulated the expression of not only Bcl-x L , but also a potent angiogenic and neurotrophic factor, vascular endothelial growth factor (VEGF) at 7 days after SCI. Conclusion: Ginsenoside Rb1 (gRb1), with putative anti-apoptotic, angiogenic and neurotrophic actions, is a candidate agent for the treatment of patients with acute SCI.

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb1 (gRb1) (C54H92O23, molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb1-induced expression of gene products responsible for neuronal death or survival, we showed that gRb1 stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-xL in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb1 treatment. Ginsenoside Rb1 appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

Background— The role of angiotensin II receptor subtypes was investigated in focal brain ischemia induced by middle cerebral artery (MCA) occlusion. Methods and Results— In Agtr2 + (wild-type) mice, MCA occlusion induced focal ischemia of ≈20% to 30% of the total area in coronal section of the brain. The ischemic area was significantly larger in angiotensin II type 2 receptor–deficient ( Agtr2 − ) mice than in Agtr2 + mice. The neurological deficit after MCA occlusion was also greater in Agtr2 − mice than in Agtr2 + mice. The decrease in surface cerebral blood flow after MCA occlusion was significantly exaggerated in the peripheral region of the MCA territory in Agtr2 − mice. Superoxide production and NADPH oxidase activity were enhanced in the ischemic area of the brain in Agtr2 − mice. An AT 1 receptor blocker, valsartan, at a nonhypotensive dose significantly inhibited the ischemic area, neurological deficit, and reduction of cerebral blood flow as well as superoxide production and NADPH oxidase activity in Agtr2 + mice. These inhibitory actions of valsartan were weaker in Agtr2 − mice. Conclusions— These results suggest that AT 2 receptor stimulation has a protective effect on ischemic brain lesions, at least partly through the modulation of cerebral blood flow and superoxide production.

Aquaporin-4 (AQP4) is located on astrocyte endfeet that face blood vessels in the brain and in the pia. It is thought to play a crucial role in the development of brain edema. To confirm the notion that sex steroids and dexamethasone influence brain edema through AQP4 regulation, we investigated the effects of 17β-estradiol, testosterone, and dexamethasone on the expression of AQP4 in cultured astrocytes. Testosterone significantly up-regulated AQP4 at the level of both protein and mRNA. At a concentration of 100 nM, testosterone significantly increased AQP4 protein levels and ameliorated the osmotic fragility of astrocytes from hypoosmotic stress, suggesting that the increased levels of AQP4 facilitated the testosterone function. Moreover, this effect was attenuated by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate, which can rapidly decrease AQP4 mRNA expression, indicating that the response was specific. These results indicate that AQP4 can alter the osmotic fragility of astrocytes and that testosterone can influence brain edema through AQP4 regulation, whereas 17β-estradiol and dexamethasone cannot. © 2003 Wiley-Liss, Inc.

The use of adenoviral vectors has recently provided a novel strategy for direct gene transfer into the cochlea. In this study, we assessed the utility of an adenoviral vector expressing glial-cell-derived neurotrophic factor (GDNF) in ischemia-reperfusion injury of the gerbil cochlea. The vector was injected through the round window 4 days before ischemic insult. The distribution of a reporter transgene was confirmed throughout the cochlea from the basal to the apical turn and Western blot analysis indicated significant upregulation of GDNF protein 11 days following virus inoculation. Hearing ability was assessed by sequentially recording compound action potentials (CAP), and the degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and Hoechst 33342. On the seventh day of ischemia, the CAP threshold shift and inner hair cell loss were remarkably suppressed in the Ad-GDNF group compared with the control group. These results suggest that adenovirus-mediated overexpression of GDNF is useful for protection against hair cell damage, which otherwise eventually occurs after transient ischemia of the cochlea.

The treatment of Alzheimer's disease (AD) remains a major challenge because of the incomplete understanding of the triggering events that lead to the selective neurodegeneration characteristic of AD brains. Here we describe a new protein, CGI-94, that is down-regulated at the mRNA level in the hippocampus of early stage AD brain. Transfection experiments with CGI-94 as a green fluorescent protein (GFP)-fusion-protein show that this protein is translocated into the nucleus of the cell. The finding that this protein, which has a bipartite nuclear localization signal, is also observed in the cytoplasm and extracellular space points to a multifunctional protein. Immunohistochemical analyses reveal that CGI-94 is mainly expressed in neurons of the hippocampal formation and the cortex but not in the cerebellar nucleus. In conclusion, the expression of the nucleolar phosphoprotein CGI-94 appears to be disturbed in early processes of neuronal degeneration.

Bone marrow mononuclear cells (BMMCs) are known to enhance recovery from ischemic insults by secreting angiogenic factors and inducing the expression of angiogenic factors from host tissues. Therefore, the transplantation of BMMCs is considered a potential approach to promoting the repair of ischemic damaged organs. Here, we investigated the influence of BMMCs on progressive hair cell degeneration after transient cochlear ischemia in gerbils. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries immediately before their entry into the transverse foramen of the cervical vertebra. An intravenous injection of BMMCs prevented ischemia-induced hair cell degeneration and ameliorated hearing impairment. A tracking study showed that BMMCs injected into the femoral vein were limited in the spiral artery of the cochlea, suggesting that, although transplanted BMMCs were retained within the spiral ganglion area of the cochlea, they were neither transdifferentiated into cochlear cells nor fused with the injured hair cells and supporting cells in the organ of Corti to restore their functions. We also showed that the protein level of neurotrophin-3 and glial cell line-derived neurotrophic factor in the organ of Corti was upregulated after treatment with BMMCs. These results suggested that BMMCs have therapeutic potential possibly through paracrine effects. Thus, we propose the use of BMMCs as a potential new therapeutic strategy for hearing loss.

We present the results of deep K � -a ndJ-band imaging of the field of a proto-cluster region near the radio galaxy 53W002 at z =2 .390 with the Subaru Telescope. The data were analyzed together with deep optical and nearinfrared (NIR) images taken with the Hubble Space Telescope to investigate the properties of ten optically compact emission-line galaxies and candidates. Excluding the three objects which may contain active galactic nuclei, many are faint or undetected at K � (λrest ≈6000 u A), and are therefore revealed to be intrinsically small starbursting objects. On the other hand, we detect few objects with colors and magnitudes expected for quiescent massive galaxies at z =2 . 4i n the f ield.

Slowly progressive degeneration of the hippocampal CA1 neurons was induced by 3-minute transient global ischemia in gerbils. Sustained degeneration of hippocampal CA1 neurons was evident 1 month after ischemia. To investigate the effects of an 18-mer peptide comprising the hydrophilic sequence of the rat saposin C domain (18MP) on this sustained neuronal degeneration, an intracerebroventricular 18MP infusion was initiated 3 days after ischemia. Histopathologic and behavior evaluations were conducted 1 week and 1 month after induction of ischemia. When compared with the vehicle infusion, 18MP treatment significantly increased the response latency time in a passive avoidance task. Increased neuronal density was also evident, as was the number of intact synapses in the hippocampal CA1 region at 1 week and 1 month after ischemia. 18MP treatment also significantly decreased the number of TUNEL-positive CA1 neurons 1 week after ischemia. Subsequent in vitro experiments using cultured neurons demonstrated that the 18MP at optimal extracellular concentrations of 1 to 100 fg/mL prevented nitric oxide–induced neuronal damage as expected and significantly up-regulated the expressions of bcl-xL mRNA and its translated protein. These results suggest that the gerbil model of 3-minute ischemia is useful in studying the pathogenesis of slowly progressive neuronal degeneration after stroke and in evaluating effects of novel therapeutic agents. It is likely that the 18MP at low extracellular concentrations prevents neuronal apoptosis possibly through up-regulation of the mitochondrial antiapoptotic factor Bcl-xL.

Recent advances in cDNA microarray technology have made it possible to analyze expression of more than 8000 genes. Using this technology, gene expression in the hippocampus containing neurofibrillary tangle-associated lesions from an Alzheimer's disease (AD) patient was compared with expression in the parietal cortex from the same patient that lacked these lesions. We also compared gene expression using a control brain. The top 20 named genes significantly up-regulated or down-regulated only in the AD brain were determined. The most up-regulated gene proved to be calcineurin Abeta mRNA (CAbeta). In situ hybridization histochemistry revealed that CAbeta was significantly up-regulated in pyramidal neurons of the hippocampus in the AD brain. RT-PCR analysis revealed that CAbeta was up-regulated in the hippocampus from two out of three AD brains while there were no changes in three control brains. Our study suggests that CAbeta may play a crucial role in the pathophysiological mechanisms in AD.

Increasing evidence suggests the presence of apoptotic cell death in many neurodegenerative diseases. However, in Binswanger's disease (BD), no information is available concerning the apoptosis-related pathologic changes that may occur in the white matter. To investigate whether apoptotic cell death is included in the pathophysiology of the white matter changes in BD, autopsied brains from patients with BD (n = 5) were compared with those of non-neurologic controls (n = 5). Terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL) was used as a marker for cell damage with DNA fragmentation. A proteolipid protein (PLP) messenger RNA (mRNA) hybridization signal was also used as a sensitive and specific marker of oligodendrocytes as well as glial fibrillary acidic protein (GFAP) immunoreactivity as a marker of astrocytes. There were frequent TUNEL-positive cells in the rarefied white matter of patients with BD. TUNEL-positive cells were found 15-fold more numerously in BD than in controls (P.01). TUNEL-positive cells were presumably oligodendrocytes because of their coexpression with PLP mRNA. The numbers of GFAP-positive astrocytes were significantly decreased in BD compared with those in control subjects. The reduction in numbers of PLP mRNA-positive oligodendrocytes were also seen in BD, but these changes did not reach the level of significance. The pathologic alterations in BD brains include increased TUNEL-positive oligodendrocytes, associated with degradation of myelin. Although TUNEL-positive glial cells did not show typical apoptotic morphologic features, these findings suggest that increase in in situ nick end labeling of oligodendrocytes in white matter may play an important role in the pathophysiology of BD.

The transcriptional factor p53 is a regulatory protein which contributes to the preservation of tissue integrity by promoting either DNA repair or apoptosis. To establish the pathophysiological role of this protein in ischemia, we produced 1 h transient middle cerebral artery (MCA) occlusion in normal and in p53-deficient mice and investigated the resulting tissue damage by multiparametric imaging. Possible genetic influences on the angioarchitecture of the MCA territory and blood flow were examined by intravascular latex infusion and laser-Doppler flowmetry. Wild-type (p53(+/+)), heterozygous (p53(+/-)) and homozygous (p53(-/-)) mice deficient for the p53 gene did not differ in respect to angioarchitecture or the effect of vascular occlusion on blood flow and general physiological parameters. Twenty-four hours after 1 h MCA occlusion, mice revealed a gene dose-dependent decline in the size of metabolic disturbances (ATP depletion and inhibition of protein synthesis) and histological injury (Cresyl Violet staining). DNA fragmentations detected by terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) did not differ in the three groups and were only present in ATP-depleted tissue. Our findings suggest that after transient focal brain ischemia p53 prevents rather than aggravates brain injury, and that this effect is brought about by mechanisms that are unrelated to the pro-apoptotic properties of this gene.

Deep near-infrared images of a blank 2'x2' section of sky near the Galactic north pole taken by Subaru Telescope are presented. The total integration times of the J and K' bands are 12.1 hours and 9.7 hours, resulting in 5-sigma limiting magnitudes of 25.1 and 23.5 mag, respectively. The numbers of sources within these limiting magnitudes found with an automated detection procedure are 385 in the J band and 350 in K'. Based on photometric measurements of these sources, we present number count vs. magnitude relations, color vs. magnitude diagrams, size vs. color relationships, etc. The slope of the galaxy number count plotted against the AB magnitude scale is about 0.23 in the 22 to 26 AB magnitude range of both bands. The spatial number density of galaxies as well as the slopes in the faint-end region given by the Subaru Deep Field (SDF) survey is consistent with those given by HST-NICMOS surveys as expressed on the AB magnitude diagram. Several sources having very large J-K' color are found including a few K' objects without detection at J. In addition, a number of faint Galactic stars are also detected, most of which are assigned to M-subdwarfs, together with a few brown dwarf candidates., 11 pages, 14 figures, to appear in Publ.Astr.Soc.Japan. The full ps file can be retrieved at ftp://ftp-cr.scphys.kyoto-u.ac.jp/pub/crmember/maihara/sdf/sdf.ps.gz

The temporospatial expression pattern of the nuclear DNA repair enzyme redox factor-1 (ref-1), the p53-activated gene (pag) 608 and the effector caspase-3 was examined by in situ hybridization histochemistry in gerbils subjected to two 10-min episodes of unilateral common carotid artery occlusion, separated by 5 h. Gene responses were correlated with the metabolic state, as revealed by regional adenosine 5′-triphosphate bioluminescent imaging, and with the degree of histological damage, as assessed by haematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated-dUTP nick end labeling (TUNEL), in order to evaluate the role of these genes in the maturation of injury. Focal infarcts developed in the dorsolateral cerebral cortex at the bregma level and the nucleus caudate–putamen within four days after repeated unilateral ischemia, as indicated by a secondary adenosine 5′-triphosphate loss after initial adenosine 5′-triphosphate recovery and by histomorphological signs of pannecrosis. The more caudal cortex at hippocampal levels and the hippocampus (CA1>CA3 area), however, exhibited selective neuronal injury without adenosine 5′-triphosphate depletion. TUNEL(+) cells appeared starting 5 h after repeated unilateral ischemia. TUNEL(+) cells reached maximum levels in the caudate–putamen at 12–24 h, but much later in the cortex and hippocampus at two days after ischemia. Remarkably few TUNEL(+) cells were noticed in the thalamus, where adenosine 5′-triphosphate state did not recover after reperfusion. Following repeated unilateral ischemia, a transient elevation of ref-1 mRNA was detected after 5 h in the cerebral cortex and hippocampal CA1 area. Ref-1 mRNA levels decreased within 12–24 h, before the onset of tissue damage. Subsequently, pag608 and caspase-3 mRNA levels increased, closely in parallel with the appearance of DNA fragmented cells, but slightly prior to the deterioration of adenosine 5′-triphosphate state. In the caudate–putamen, pag608 and caspase-3 mRNAs reached maximum levels already 12–24 h after repeated common carotid artery occlusion, when DNA fragmentation was most prominent, and declined thereafter. In the cortex and hippocampal CA1-3 areas, where DNA damage appeared more slowly, pag608 and caspase-3 mRNAs were induced starting 24 h after ischemia, and remained elevated even after two to four days. The levels of pag608 and caspase-3 mRNAs were similar at rostral and caudal levels of the cortex, as well as in the hippocampal CA1 and CA3 area, although the degree of injury differed considerably between these structures. Notably, pag608 and caspase-3 mRNAs were not elevated in the thalamus after repeated unilateral ischemia. The present report shows a close temporal association between the induction of ref-1, pag608 and caspase-3 mRNAs, the manifestation of cell injury and the secondary adenosine 5′-triphosphate depletion in infarcting brain areas, suggesting (i) that de novo responses of these genes may be involved in the maturation of cell injury and (ii) that apoptotic programs and the secondary deterioration of cerebral energy state may interfere with each other after ischemia.

We construct infrared star count models of the Galaxy applicable at faint magnitudes. In addition to dwarfs and subdwarfs, we also take into account L dwarfs, T dwarfs, and white dwarfs of both disk and halo. As a test case of the models, we analyze the infrared imaging data at J and K' obtained during the Subaru Deep Field Survey to study stellar objects. Infrared star count models we have obtained predict that the Next Generation Space Telescope will primarily see T dwarfs, M subdwarfs, and old halo white dwarfs at faint magnitudes., 20 pages, 3 figures. To be published in the November 2000 issue of the Astronomical Journal

To elucidate the mechanism of neuronal death in Alzheimer's disease, we investigated the effects of overexpression of wild-type Alzheimer amyloid precursor protein (APP) on neuronal cells and glial cells in vivo. When an APP695-expressing adenovirus was injected into the dorsal hippocampal region, a number of neurons in remote areas were positively stained with anti-APP monoclonal antibody, and underwent severe degeneration from 3 to 7 days after viral inoculation. Most degenerating neurons were immunopositive with both APP and activated caspase-3, but some neurons that expressed activated caspase-3 were not expressing APP from 7 to 14 days after virus injection. In the neighborhood of the degenerating neurons, activated microglia/macrophages, which were identified by the phenotypic marker C3bi receptor (CD11b/c; OX-42), were observed, and some of them appeared to phagocytose the caspase-3-immunopositive degenerating neurons. In addition to microglia/macrophages, infiltrating leukocytes expressing CD45 or CD4 were also detected. These results suggest that the increased accumulation of APP induced not only caspase-3-mediated death machinery, but also inflammatory responses including microglial activation. These inflammatory responses might cause further neurodegeneration through the alternative pathway that might activate the caspase-3-mediated death machinery without APP expression.

We present the results of the near-infrared spectroscopy of the Pluto-Charon system, conducted in the K band at two different sub-Earth longitudes. The spatially resolved images resulted in separated spectra with a spectral resolution of approximately 530. The spectrum of Charon showed a broad absorption feature at 2 /im during both orbital phases, suggesting a uniform distribution of water ice. In the separated spectrum of Pluto obtained in June, we confirmed the absorption bands of solid methane, carbon monoxide, and nitrogen ices. Additional weak dips have been found at 2.28, 2.32, and 2.40 /xm in the spectrum of the opposite side observed in May. In order to examine the carrier of these dips, we performed model calculations of the reflectance of Pluto using Hapke's bidirectional reflectance model for icy intimate mixtures. By adding a small amount of solid ethane into the mixture, we successfully reproduced the dips at 2.28 and 2.315 /im, but the absorption at 2.40 /xm is more distinct than that in the observed spectrum.

We have investigated the optical and near-infrared colors of K'-selected galaxies in clusters at z ~ 1.2 near to the radio galaxy 3C 324 using images obtained with the Subaru telescope and archival HST data. The distribution of colors of the galaxies in the cluster region is found to be fairly broad, and it may imply significant scatter in their star-formation histories, although the effect of contamination of field galaxies is uncertain. The red sequence of galaxies whose R-K colors are consistent with passive evolution models for old galaxies is found to be truncated at K' ~ 20 mag, and there are few fainter galaxies with similar red colors in the cluster region. We find that the bulge-dominated galaxies selected by quantitative morphological classification form a broad sequence in the color-magnitude diagram, whose slope is much steeper than that expected from metallicity variations within a passively evolving coeval galaxy population. We argue that the observed color-magnitude sequence can be explained by metallicity and age variations, and the fainter galaxies with K' > 20 mag may be 1-2 Gyr younger than the brighter galaxies. Some spatial segregation of the color and K'-band luminosity is seen in the sky distribution; the redder and the brighter objects tend to be located near 3C 324., 16 pages, 10 figures, accepted for publication in PASJ

We present high spatial resolution images of the gravitational-lens system PG 1115+080 taken with the near-infrared camera (CISCO) on the Subaru telescope. The FWHM of the combined image is $0.''32$ in the $K'$-band, yielding spatial resolution of $0.''14$ after a deconvolution procedure. This is a first detection of an extended emission adjacent to the A1/A2 components, indicating the presence of a fairly bright emission region with a characteristic angular radius of $\sim$ 5 mas (40 pc). The near-infrared image of the Einstein ring was extracted in both the $J$ and $K'$ bands. The $J-K'$ color is found to be significantly redder than that of a synthetic model galaxy with an age of 3 Gyr, the age of the universe at the quasar redshift., 11 pages, 6 figures. Accepted for publication in PASJ(2000)

We present the 2.12~$\mu$m narrow-band image of the Hubble Deep Field North taken with the near-infrared camera (CISCO) on the Subaru telescope. Among five targets whose H$\alpha$ or [O~{\sc iii}] emission lines are redshifted into our narrow-band range expected from their spectroscopic redshift, four of them have strong emission lines, especially for the two [O~{\sc iii}] emission-line objects. The remaining one target shows no H$\alpha$ emission in spite of its bright rest-UV luminosity, indicating that this object is already under the post-starburst phase. The volume-averaged $SFR$ derived from the detected two H$\alpha$ emission is roughly consistent with that evaluated from the rest-UV continuum., Comment: 12 pages, 7 figures. Accepted for publication in PASJ(2000)

Abnormalities in the homeostasis of the renin-angiotensin system have been implicated in the pathogenesis of vascular disorders, including stroke. The authors investigated whether angiotensinogen (AGN) knockout mice exhibit differences in brain susceptibility to focal ischemia, and whether such differences can be related to special features of the collateral circulation. Wild-type and AGN-knockout mice were submitted to permanent suture occlusion of the middle cerebral artery (MCA). The collateral vascular system was visualized by systemic latex infusion, and the ischemic lesions were identified by cresyl-violet staining. The core and penumbra of the evolving infarct were differentiated by bioluminescence and autoradiographic imaging of A TP and protein biosynthesis, respectively. In wild-type mice, mean arterial blood pressure was 95.0 ± 8.6 mm Hg, and the diameter of fully relaxed anastomotic vessels between the peripheral branches of the anterior and middle cerebral arteries 26.6 ± 4.0 μm In AGN knockouts, mean arterial blood pressure was significantly lower, 71.5 ± 8.5 mm Hg ( P

C57Black/6 and SV129 mice are widely used for the production of transgenic mutants in molecular stroke research but the ischemic susceptibility of these strains is influenced by differences in vascular anatomy and the responsiveness to excitotoxins and vasodilatory stimuli. To differentiate between these opposing effects on infarct size, the vascular territory of the two strains was correlated with the hemodynamic, metabolic, and morphological consequences of permanent middle cerebral artery (MCA) occlusion. The vascular anatomy was studied by latex infusion, brain infarction by vital staining, the size of the ischemic penumbra by imaging of ATP and protein synthesis, and blood flow by laser–Doppler flowmetry. In C57Black/6 mice the MCA-supplied vascular territory and the size of brain infarcts were significantly larger than in SV129 mice but the size of the penumbra and the residual blood flow in the center of the MCA-supplying territory were similar in both strains. These findings suggest that differences in infarct size in C57Black/6 and SV129 mice are determined mainly by the vascular anatomy and not by differences in collateral vascular responsiveness or excitotoxicity.

Neuronal death after brain ischemia is mainly due to necrosis but there is also evidence for involvement of apoptosis. To test the importance of apoptosis, we investigated the effect of targeted disruption of the apoptosis-suppressive gene bcl-2 on the severity of ischemic brain injury. Transient focal ischemia for 1 hour was induced by occlusion of the middle cerebral artery in homozygous (n=7) and heterozygous (n = 6) bcl-2 knockout mice as well as in their wildtype littermates (n=5). Bcl-2 ablation did not influence cerebral blood flow but it significantly increased infarct size and neurological deficit score at 1 day after reperfusion in a gene-dose dependent manner. The exacerbation of tissue damage in the absence of Bcl-2 underscores the importance of apoptotic pathways for the manifestation of ischemic injury after transient vascular occlusion.

The Zac1 gene encodes a zinc finger protein that regulates both apoptosis and cell cycle arrest in vitro. Furthermore, Zac1 protein seems to trans-activate the gene encoding the type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP). Northern blot analysis revealed high levels of Zac1 mRNA in the rodent brain. In the present study, we demonstrate by in situ hybridization histochemistry a progressive increase in Zac1 transcripts in the mouse brain from day 1 to day 3 following transient focal cerebral ischemia. Moreover, we observed an up-regulation of PACAP type I receptor mRNA expression showing a similar temporospatial distribution. Late induction of cell death promoting Zac1 in the post-ischemic brain may be attributed to delayed or secondary cell death. Co-induction of the type I receptor for neurotrophic PACAP however, points to a role in restorative processes.

A reproducible model of thread occlusion of the middle cerebral artery (MCA) was established in C57 Black/6J mice by matching the diameter of the thread to the weight of the animals. For this purpose, threads of different diameter (80 to 260 μm) were inserted into the MCA of animals of different weights (18 to 33 g), and the success of vascular occlusion was evaluated by imaging the ischemic territory on serial brain sections with carbon black. Successful occlusion of the MCA resulted in a linear relationship between body weight and thread diameter ( r = 0.46, P < 0.01), allowing precise selection of the appropriate thread size. Laser-Doppler measurements of CBF, neurological scoring, and 2,3,5-triphenyltetrazolium chloride staining confirmed that matching of animal weight and suture diameter produced consistent cerebral infarction. Three hours after MCA occlusion, imaging of ATP, tissue pH, and cerebral protein synthesis allowed differentiation between the central infarct core, in which ATP was depleted, and a peripheral penumbra with reduced protein synthesis and tissue acidosis but preserved ATP content. Perfusion deficits and ischemic tissue alterations could also be detected by perfusion- and diffusion-weighted magnetic resonance imaging, demonstrating the feasibility of dynamic evaluations of infarct evolution. The use of multiparametric imaging techniques in this improved MCA occlusion model opens the way for advanced pathophysiological studies of stroke in gene-manipulated animals.

To evaluate the reversibility of neural function in the brainstem following ischemia, we investigated the effect of transient brainstem ischemia on the brainstem auditory evoked potential in gerbils. Brainstem ischemia was produced by bilateral extracranial occlusion of vertebral arteries. Local cerebral blood flow was measured by quantitative autoradiography after 5 min of ischemia and was reduced to less than 3 ml/100 g per min in the pons and lower midbrain, indicating severe and reproducible brainstem ischemia. During brainstem ischemia, brainstem auditory evoked potential waveforms disappeared completely. After a brief ischemic insult (5 min), all four brainstem auditory evoked potential components recovered to normal. After longer ischemic insults (10-30 min), brainstem auditory evoked potential components never recovered to normal. Microtubule-associated protein 2 immunoreactivity revealed differential vulnerability of the acoustic relay nuclei in the brainstem. Neurons in the lateral lemniscus were most vulnerable, followed in order by neurons in the trapezoid body, the superior olive and the cochlear nucleus. We also demonstrated a close relationship between the reversibility of ischemia-induced changes on brainstem auditory evoked potential and ischemic lesions of these relay nuclei. These data may be useful for evaluating the therapeutic window of thrombolytic therapy during acute vertebrobasilar occlusion.

To assess whether ischemia could induce GAP-43 mRNA expression, we performed in situ hybridization in gerbil brains that had been subjected to 5 min of global ischemia. In control dentate granule cells, little hybridization was detected in contrast to the intense signal generated by pyramidal neurons of the adult hippocampal formation. After ischemia, we detected a robust GAP-43 signal over hippocampal granule cells at 3 h of reperfusion, persisting through 7 days, and disappearing by 14 days. This demonstrated GAP-43 gene induction after ischemia, and suggests that GAP-43 may be involved in reactive events, including fiber sprouting and synaptic reorganization, that follow ischemia.

Fas antigen mRNA induction in the brain was examined using a transient global cerebral ischemia model in BALB/C mice. Northern blot analysis revealed little Fas antigen mRNA expression in the brains of sham-operated mice. A marked induction of Fas mRNA expression was detected in the brains of mice 6 h after 30 min of cerebral ischemia. These results suggest a possible apoptotic mechanism for cell death, mediated by the Fas antigen, in postischemic brain.

A new gerbil model of hindbrain ischemia was induced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Carbon black studies, performed at 5 min after occlusion, revealed that the pons-medulla oblongata, and the cerebellum were quite ischemic in all animals. Cardiovascular changes in mean arterial blood pressure (MABP) and heart rate were recorded until 30 min after occlusion, and revealed that the typical cerebral ischemic response (i.e., abrupt increase in MABP, bradycardia, and apnea) was elicited in all animals (n = 10). Thirty minutes after occlusion, animals (n = 4) were decapitated and immersion-fixed. Brain sections were stained with hematoxylin-eosin (HE) and also immunostained for microtubule-associated protein 2 in order to evaluate ischemic neuronal damage from 30 min of ischemia. By HE staining, ischemic lesions were detected bilaterally in the oculomotor, the trigeminal motor, the lateral vestibular, and the cerebellar interpositus nucleus. In addition, immunostaining revealed ischemic lesions in several other hindbrain areas. In conclusion, we could successfully establish a new gerbil model of hindbrain ischemia. Carbon black perfusion and hemodynamic studies revealed that severe and reproducible hindbrain ischemia was produced. By histopathological examination, we could also clearly demonstrate symmetrical ischemic lesions in several hindbrain areas.

Differential vulnerability in the hindbrain neurons was examined immunohistochemically during hindbrain ischemia in the gerbil. Hindbrain ischemia was produced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Local cerebral blood flow was measured by quantitative autoradiographic technique after 5 min of ischemia and was reduced to less than 5 ml/100 g per min in the cerebellum, the pons, and the medulla, indicating that severe and reproducible hindbrain ischemia was induced immediately after occlusion. For immunohistochemical investigation, four gerbils each were used for each ischemie period of 5, 10, 15, and 30 min. Immunohistochemical lesions, detected by the reaction for microtubule-associated protein 2, were visible in the lateral vestibular nucleus and the cerebellar interpositus nucleus even after 5 min of ischemia. These results suggested that these areas were more vulnerable than others, although blood flow was markedly reduced in various regions of the hindbrain. In contrast, areas related to respiratory or cardiovascular control were rather resistant to ischemia. The present study suggests that selective vulnerability during hindbrain ischemia depends mainly on different metabolic characteristics inherent to various neurons in the hindbrain.

Brief periods of non-lethal cerebral ischemia can induce resistance against subsequent lethal ischemia. In this study, asymptomatic gerbils after unilateral carotid artery ligation were subjected to 5 min of forebrain ischemia. The prolonged but mild hypoperfusion, by carotid occlusion, induced susceptibility at 1 day and tolerance at 30 days to lethal ischemia in the hippocampal neurons. The neuroprotective effect correlated well with induction of heat shock protein 72 in the hippocampal neurons. These results suggested that neuronal cells possess a cellular response to sublethal hypoperfusion and can survive forthcoming ischemic stress.

Background Because of the lack of reproducible brainstem ischemia models in rodents, the temporal profile of ischemic lesions in the brainstem after transient brainstem ischemia has not been evaluated intensively. Previously, we produced a reproducible brainstem ischemia model of Mongolian gerbils. Here, we showed the temporal profile of ischemic lesions after transient brainstem ischemia. Results Brainstem ischemia was produced by occlusion of the bilateral vertebral arteries just before their entry into the transverse foramina of the cervical vertebrae of Mongolian gerbils. Animals were subjected to brainstem ischemia for 15 min, and then reperfused for 0 d (just after ischemia), 1 d, 3 d and 7 d (n = 4 in each group). Sham-operated animals (n = 4) were used as control. After deep anesthesia, the gerbils were perfused with fixative for immunohistochemical investigation. Ischemic lesions were detected by immunostaining for microtubule-associated protein 2 (MAP2). Just after 15-min brainstem ischemia, ischemic lesions were detected in the lateral vestibular nucleus and the ventral part of the spinal trigeminal nucleus, and these ischemic lesions disappeared one day after reperfusion in all animals examined. However, 3 days and 7 days after reperfusion, ischemic lesions appeared again and clusters of ionized calcium-binding adapter molecule-1(IBA-1)-positive cells were detected in the same areas in all animals. Conclusion These results suggest that delayed neuronal cell death took place in the brainstem after transient brainstem ischemia in gerbils.

Although signal transducer and activator of transcription 3 (Stat3) plays crucial roles in the determination of neural stem cell (NSC) fate, Stat3 has multiple roles in NSC function. Moreover, Stat3 plays important roles in neuronal survival and tumorigenesis. To investigate the overall effects of Stat3 on NSC fate, NSC were isolated from Stat3(flox/flox) mouse embryos (E14-15d), in which both Stat3 alleles are flanked by LoxP sites. Isolated NSC was inoculated with an adenovirus vector expressing Cre recombinase (Ad.nCre) or a control adenovirus vector expressing beta-galactosidase (Ad.nLz). Three days later, quantitative real-time PCR (qPCR) analysis revealed that treatment with Ad.nCre eliminated stat3 mRNA expression in NSC. Promoter assay confirmed that overexpression of nCre inhibited transactivation of acute responsive element (APRE) and blocked Stat3 function in NSC. Moreover, Western blot analysis and immunocytochemical analysis revealed that elimination of Stat3 in NSC promoted neurogenesis and inhibited astrogliogenesis. In addition, we investigated the effects of Stat3 elimination in NSC on the mRNA expression of Notch family members and bHLH factors. Consequently, qPCR analysis showed that elimination of Stat3 in NSC promoted neurogenesis and inhibited astrogliogenesis through down-regulation of notch1, notch2 and hes5, but not hes1 mRNA expression.

Ischemic tolerance in the cochlea was investigated in a gerbil model of cochlear ischemia. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries. The gerbils were divided into two groups; single ischemia group and double ischemia group. In the single ischemia group, animals were subjected to lethal cochlear ischemia for 15 min. In the double ischemia group, animals were subjected to sublethal cochlear ischemia for 2 min at 2 days before lethal ischemia for 15 min. Consequently, sublethal ischemia prevented lethal ischemia-induced hair cell degeneration and ameliorated hearing impairment, suggesting ischemic tolerance in the cochlea.

The differentiation and proliferation of neural stem cells (NSCs) are regulated by a combination of their intrinsic properties (e.g., transcription factors, epigenetic factors, and microRNA regulation) and cell-extrinsic properties from the microenvironment around NSC (e.g., cytokines, growth factors, and cell-cell contact). Recently, there has been a great interest in clarifying the mechanism of the influence of the microenvironment on NSCs, especially cell-cell contact between NSCs and other types of cells nearby. In this study, we investigated whether microglial (Mi) cells influence the fate of NSCs. Coculture study showed that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs through their paracrine effects. This microglia-induced astrogliogenesis was inhibited by AG490 and by overexpression of the dominant-negative form of Stat3 and SOCS3. Promoter assay revealed transactivation of Stat3 function in NSCs by Mi cells. Gene expression study revealed that mRNA of Notch family members (notch1-3) and sox9 in NSCs was significantly upregulated by Mi cells, and this up-regulation was inhibited by AG490. These results demonstrated that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs by activating Stat3 function and via notch and sox9 signaling pathways.