Biomedical subjects

Norio Takagi

Publications and source records attributed to Norio Takagi.

At least 19 recordsLinked to original sources

PubMed2006

Hepatocyte growth factor attenuates cerebral ischemia-induced increase in permeability of the blood-brain barrier and decreases in expression of tight junctional proteins in cerebral vessels.

Hepatocyte growth factor (HGF) exerts its physiological activities as that of an organotropic factor for regeneration and can prevent ischemia-induced injuries; however, its effect and mechanism of action under in vivo pathophysiological conditions remains to be determined. Recently, we demonstrated that treatment with human recombinant HGF (hrHGF) attenuated the disruption of the blood-brain barrier (BBB) observed after microsphere embolism-induced sustained cerebral ischemia. To see if tight junctional proteins were involved in this attenuation, in the present study, we investigated the effects of HGF on the levels of occludin and zonula occludens (ZO)-1 in cerebrovascular endothelial cells after microsphere embolism. Sustained cerebral ischemia was induced by the injection of 700 microspheres (48 microm diameter) into the right internal carotid artery of rats. hrHGF was injected into the right ventricle of the brain by using an osmotic pump at a dose of 30 microg/7 days per animal. The levels of tight junctional proteins in the endothelial cells were examined by immunohistochemical analysis. Treatment with hrHGF attenuated the decrease in the expression of occludin and ZO-1 proteins in the endothelial cells that occurred after sustained cerebral ischemia. Furthermore, treatment with hrHGF resulted in retention of these tight junctional proteins in fluorescein isothiocyanate (FITC)-albumin-perfused cerebral vessels, which did not leak FITC-albumin in the ipsilateral cortex. These results suggest that HGF-mediated maintenance of the tight junctional proteins in the endothelial cells may be a possible mechanism for the protective effect of HGF against the disruption of the BBB after cerebral ischemia.

Animals↗

PubMed2006

The protective effect of hepatocyte growth factor against cell death in the hippocampus after transient forebrain ischemia is related to the improvement of apurinic/apyrimidinic endonuclease/redox factor-1 level and inhibition of NADPH oxidase activity.

Early oxidative DNA damage is regarded to be an initiator of neuronal apoptotic cell death after cerebral ischemia. Although evidence suggests that HGF has the ability to protect cells from oxidative stress, it remains unclear as to how HGF suppresses oxidative DNA damage after cerebral ischemia. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) is a multifunctional protein in the DNA base repair pathway that is responsible for repairing apurinic/apyrimidinic sites in DNA after oxidation. We demonstrated that both the immunoreactivity and the number of APE/Ref-1-positive cells in the hippocampal CA1 region were decreased after transient forebrain ischemia and that treatment with HGF suppressed this reduction. The expression of Cu/ZnSOD and MnSOD in the hippocampal CA1 region did not change after ischemia, regardless of treatment with or not with HGF. The activity of NADPH oxidase was increased mainly in glia-like cells in the hippocampal CA1 region after ischemia, and this increase was attenuated by HGF treatment. These results suggest that the protective effects of HGF against cerebral ischemia-induced cell death in the hippocampal CA1 region are related to the improvement of neuronal APE/Ref-1 expression and the inhibition of NADPH oxidase activity in glia-like cells.

Apoptosis↗

PubMed2006

Effects of hepatocyte growth factor on phosphorylation of extracellular signal-regulated kinase and hippocampal cell death in rats with transient forebrain ischemia.

Hepatocyte growth factor (HGF) has been implicated in protection against several types of cell injuries. We investigated the effects of human recombinant HGF (hrHGF) on the selective neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in rats and explored the nature of the intracellular signaling pathway for the protection against this neuronal injury. hrHGF was injected continuously into the hippocampal CA1 region directly using an osmotic pump from 10 min to 72 h after the start of reperfusion. The marked increase in the number of TUNEL-positive cells found in the CA1 region after ischemia was almost completely abolished by the hrHGF treatment. Akt phosphorylation as well as IkappaB phosphorylation, which has been implicated in events downstream of the Akt, was not affected by hrHGF treatment. Extracellular signal-regulated kinase (ERK) phosphorylation was decreased in the CA1 region with time after ischemia. hrHGF increased or recovered ERK phosphorylation without changing the total amount of ERK protein. Immunohistochemical analysis demonstrated that phosphorylated ERK was colocalized with a neuronal nucleus marker NeuN in the hippocampal CA1 region of ischemic rats with hrHGF treatment at the early period after reperfusion. These results suggest that the protective effects of hrHGF against neuronal death in the hippocampal CA1 after transient forebrain ischemia could be related to an ERK-dependent pathway.

Animals↗

PubMed2006

Prevention of apoptosis-inducing factor translocation is a possible mechanism for protective effects of hepatocyte growth factor against neuronal cell death in the hippocampus after transient forebrain ischemia.

Hepatocyte growth factor (HGF) is one of the prospective agents for therapy against a variety of neurologic and neurodegenerative disorders, although the precise mechanisms for the effect of HGF remain to be elucidated. We showed that treatment with HGF protected hippocampal cornu ammonis (CA) subregion 1 neurons from apoptotic cell death after transient forebrain ischemia. Accumulating evidence indicates that ischemia-induced neuronal damage occurs via caspase-independent pathways. In the present study, we focused on the localization of apoptosis-inducing factor (AIF), which is an important protein in the signal-transduction system through caspase-independent pathways, to investigate the possible mechanism for the protective effect of HGF after transient forebrain ischemia. Hepatocyte growth factor attenuated the increase in the expression of AIF protein in the nucleus after transient forebrain ischemia. We further explored the upstream components of AIF translocation. Primary DNA damage induced by Ca(2+) influx and subsequent NO formation are thought to be the initial events for AIF translocation, which results in the subsequent DNA damage by AIF. Hepatocyte growth factor prevented the primary oxidative DNA damage, as was estimated by using anti-8-OHdG (8-hydroxy-2'-deoxyguanosine) antibody. Oxidative DNA damage after ischemia is known to lead to the activation of poly(ADP-ribose) polymerase (PARP) and p53, resulting in AIF translocation. Marked increases in the PAR polymer formation and the expression of p53 protein after ischemia were effectively prevented by HGF treatment. In the present study, we first showed that HGF was capable of preventing neuronal cell death by inhibiting the primary oxidative DNA damage and then preventing the activation of the PARP/p53/AIF pathway.

Animals↗

PubMed2005

Cerebral ischemia enhances tyrosine phosphorylation of occludin in brain capillaries.

Cerebral ischemia induces disruption of the blood-brain barrier (BBB), and this disruption can initiate the development of brain injuries. Although the molecular structure of tight junctional complexes in the BBB has been identified, little is known about alterations of tight junctional proteins after cerebral ischemia. Therefore, we investigated alterations of tight junctional proteins, i.e., occludin and zonula occludens (ZO)-1, in isolated rat brain capillaries after microsphere-induced cerebral embolism. We demonstrated that the levels of occludin and ZO-1 had decreased after the embolism. The embolism also resulted in a marked increase in tyrosine phosphorylation of occludin, which was coincident with an increase in the activity of c-Src. These results suggest that a decrease in the levels of occludin and ZO-1, and an increase in tyrosine phosphorylation of occludin may play an important role in the disruption of tight junctions, which may lead to dysfunction of the BBB after cerebral ischemia.

Animals↗

PubMed2005

Inhibition of apoptosis-inducing factor translocation is involved in protective effects of hepatocyte growth factor against excitotoxic cell death in cultured hippocampal neurons.

Although hepatocyte growth factor (HGF) and its receptor are expressed in various regions of the brain, their effects and mechanism of action under pathological conditions remain to be determined. Over-activation of the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor, has been implicated in a variety of neurological and neurodegenerative disorders. We investigated the effects of HGF on the NMDA-induced cell death in cultured hippocampal neurons and sought to explore their mechanisms. NMDA-induced cell death and increase in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were prevented by HGF treatment. Although neither the total amounts nor the mitochondrial localization of Bax, Bcl-2 and Bcl-xL were affected, caspase 3 activity was increased after NMDA exposure. Treatment with HGF partially prevented this NMDA-induced activation of caspase 3. Although the amount of apoptosis-inducing factor (AIF) was not altered, translocation of AIF into the nucleus was detected after NMDA exposure. This NMDA-induced AIF translocation was reduced by treatment with HGF. In addition, increased poly(ADP-ribose) polymer formation after NMDA exposure was attenuated by treatment with HGF. These results suggest that the protective effects of HGF against NMDA-induced neurotoxicity are mediated via the partial prevention of caspase 3 activity and the inhibition of AIF translocation to the nucleus.

Animals↗

PubMed2005

Changes in the expression of Hes5 and Mash1 mRNA in the adult rat dentate gyrus after transient forebrain ischemia.

Accumulating evidence indicates that neurogenesis in the adult brain occurs in restricted brain regions, including the hippocampal dentate gyrus and is promoted by ischemia. The mechanism responsible for ischemia-induced neurogenesis in the adult brain, however, remains unclear. Notch pathway plays a pivotal role in the regulation of the timing for differentiation and determination of the fate of neural progenitor cells in the developing nervous system. To elucidate the mechanism underlying ischemia-induced neurogenesis, we investigated changes in the expression of mRNAs of Hes5, which is a downstream target of Notch, and Mash1, a neurogenic basic helix-loop-helix factor, which is negatively regulated by Hes5, in the adult hippocampal dentate gyrus after transient forebrain ischemia. Transient forebrain ischemia was produced by four-vessel occlusion procedure in rats. The levels of Hes5 mRNA decreased on days 1 and 3 after the start of reperfusion and the decreased levels of the mRNA returned to the basal level by 5 days after ischemia. In contrast, the level of Mash1 mRNA increased on day 1 and then returned to the basal level by 3 days after ischemia. These results suggest that an inhibition of Notch activity and subsequent expression of neurogenic basic helix-loop-helix factors, including Mash1, may, at least in part, contribute to ischemia-induced neurogenesis in the adult dentate gyrus.

Animals↗

PubMed2005

Time course and sequence of pathological changes in the cerebellum of microsphere-embolized rats.

Ischemia is a major cause of damage to the central nervous system as a consequence of stroke or trauma. Here, we analyzed with high temporal resolution the time course of pathological changes in the neurons (granule and Purkinje cells) and glia (Bergmann and astroglia cells) in the cerebellar cortex and white matter. The period studied ranged from 30 min to 7 days after a microsphere-induced embolism used as a model of stroke and multi-infarct dementia. Some pathological changes in the neurons in the cerebellar cortex were identified early, that is, beginning at 3 h after the microsphere-induced embolism, and glial pathology appeared only later. The pathological changes in the white matter also appeared slightly later, that is, 6 h after embolism and were less pronounced than those in the cerebellar cortex. This suggests that neuronal pathology is induced more rapidly and/or more easily than the glial pathology. In addition, BrdU staining shows that cell proliferation is limited to a 1-day period beginning about 1 day after the embolism. These data demonstrate that changes after an ischemic lesion of the cerebellum proceeds from upper cerebellar cortex to deeper cerebellar cortex or white matter and also that microsphere-induced changes proceed from neuronal to glial pathology.

Animals↗

PubMed2004

Effects of nefiracetam on the levels of brain-derived neurotrophic factor and synapsin I mRNA and protein in the hippocampus of microsphere-embolized rats.

Our recent study demonstrated that nefiracetam, N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, prevented impairment of the cyclic AMP (cAMP)/cAMP-responsive element binding (CREB) protein signaling pathway in sustained cerebral ischemia. The purpose of the present study was to determine whether nefiracetam has an effect on the expression of brain-derived neurotrophic factor (BDNF) and synapsin I mRNAs that are believed to be produced via CREB, and the alteration in their protein contents in the hippocampus after cerebral ischemia. Sustained cerebral ischemia was induced by injection of 700 microspheres into the right hemisphere of each rat. The rats were treated once daily with 10 mg/kg nefiracetam, p.o., from 15 h after the operation. Treatment with nefiracetam reduced the prolongation of the escape latency in the water maze test on days 7-9 after microsphere embolism-induced sustained cerebral ischemia, suggesting an improvement in the spatial learning function. Microsphere-embolized rats on day 5 showed decreases in BDNF and synapsin I mRNA levels and their protein contents in the ipsilateral hippocampus. Treatment with nefiracetam partially attenuated the decreases. These results suggest that enhancement of BDNF and synapsin I expression by nefiracetam treatment may be, at least in part, due to the improvement in the CREB binding activity, contributing to the prevention of learning and memory dysfunction after sustained cerebral ischemia.

Animals↗

PubMed2004

Hepatocyte growth factor improved learning and memory dysfunction of microsphere-embolized rats.

Hepatocyte growth factor (HGF), an organotropic factor for regeneration and protection in various organs, has the ability to attenuate cerebral ischemia-induced cell death. The effect of HGF on learning and memory function after cerebral ischemia, however, remains unknown. We have demonstrated that administration of human recombinant HGF (hrHGF) into the ventricle reduced prolongation of the escape latency in acquisition and retention tests of the water maze task on Days 12-28 after microsphere embolism-induced cerebral ischemia. Treatment with hrHGF also attenuated the decrease in viable area and the density and number of perfused cerebral vessels, particularly those with a diameter smaller than 10 microm, of the ipsilateral hemisphere on Day 28 after the cerebral ischemia. We observed that treatment with hrHGF reduced the number of TUNEL-positive cerebral endothelial cells at the early stage after the ischemia. These results suggest that hrHGF prevents learning and memory dysfunction seen after sustained cerebral ischemia by protecting against injury to the endothelial cells. HGF treatment may be a potent therapeutic strategy for cerebrovascular diseases, including cerebral infarct and vascular dementia.

Analysis of Variance↗

PubMed2004

Hepatocyte growth factor attenuates cerebral ischemia-induced learning dysfunction.

Hepatocyte growth factor (HGF) acts as an organotropic factor for regeneration and protection in various organs and has the ability to attenuate cerebral ischemia-induced cell death. However, the effect of HGF on learning and memory function after a cerebral ischemic event is unknown. We demonstrate here that administration of human recombinant HGF (hrHGF) into the ventricle reduced the prolongation of the escape latency in the acquisition and retention tests in the water maze task on days 12-28 after microsphere embolism-induced cerebral ischemia. In addition, disruption of the blood-brain barrier at the early stage after microsphere embolism, which was determined by FITC-albumin leakage, was markedly reduced by treatment with hrHGF. We demonstrated that this effect of hrHGF on the blood-brain barrier was associated with protection against the apoptotic death of the cerebral endothelial cells at the early stage after the ischemia. These results suggest that hrHGF can prevent the learning and memory dysfunction soon after sustained cerebral ischemia by protecting against injury to the endothelial cells. The use of HGF may be a potent strategy for the treatment of cerebrovascular diseases, including cerebral infarct and vascular dementia.

Animals↗

PubMed2004

Altered Bad localization and interaction between Bad and Bcl-xL in the hippocampus after transient global ischemia.

Accumulating evidence indicates that the mitochondrial cell-death pathway, which involves the release of cytochrome c from mitochondria, participates in neuronal cell death after transient cerebral ischemia. However, the upstream events, that induce cytochrome c release after transient global ischemia are not fully understood. Bad is a pro-apoptotic member of the bcl-2 gene family that promotes apoptosis by binding to and inhibiting functions of anti-apoptotic proteins Bcl-2 and Bcl-xL. We investigated the effects of transient (15 min) global ischemia on the intracellular localization of Bad and the interaction of Bad with calcineurin, Akt or Bcl-xL in the vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Immunoblotting analysis revealed that the amount of Bad in mitochondria significantly increased after ischemia. Co-immunoprecipitation studies showed decreased interactions of Bad with Akt and calcineurin in the cytosol and increased binding with Bcl-xL in the mitochondrial fraction of hippocampal CA1, but not in the CA3/dentate gyrus region. Further, we examined the effect of recombinant Bad on the cytochrome c release from isolated mitochondria. Treatment with both recombinant Bad and calcium, but not with recombinant Bad alone, induced cytochrome c release. These results suggest that changes in localization and complex formation by Bad are, at least in part, involved in the vulnerability of cells after transient global ischemia.

Animals↗

PubMed2004

The effects of monobromobimane on neuronal cell death in the hippocampus after transient global cerebral ischemia in rats.

Calcium accumulation and free radical formation in the mitochondria are suggested to result in opening of the mitochondrial permeability transition pore that may be an initial step in neuronal cell death. The purpose of the present study was to determine whether monobromobimane (MBM) was a possible protective agent against neuronal cell death after transient global ischemia and the swelling of isolated hippocampal mitochondria. Infusion of MBM (1 or 3 microg) to cerebral ventricles 30 min before ischemia attenuated the expression of TUNEL-labeled cells and neuronal cell death in the hippocampal CA1 region at 72 h of reperfusion dose-dependently. Treatment with MBM inhibited an increase in caspase-3-like activity at 48 h of reperfusion in the hippocampus. MBM (30-300 microM) also inhibited an enhanced swelling rate induced by Ca2+ and phenylarsineoxide in the isolated hippocampal mitochondria. These results suggest that in vivo treatment with MBM may protect against neuronal cell death through inhibition of the mitochondrial swelling and caspase-3-dependent apoptotic pathway.

Animals↗

PubMed2004

Phosphorylation of extracellular-regulating kinase in NMDA receptor antagonist-induced newly generated neurons in the adult rat dentate gyrus.

Neurogenesis in the adult brain is promoted by various stimulations. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus. There is no agreed conclusion, however, as to whether newly generated neurons after NMDA receptor blockade obtain functional properties. We investigated the functional maturation of newly generated neurons after NMDA receptor blockade. In the dentate gyrus, 80% of newly generated cells differentiated into the phenotype of mature neurons at 29 days after the single intraperitoneal injection of an NMDA receptor antagonist MK-801. The number of newly generated neurons after MK-801 treatment was significantly greater than that in the saline-treated group. The neurogenic basic helix-loop-helix transcription factor NeuroD protein in the dentate gyrus after MK-801 treatment was expressed transiently in proliferative cells, but not in mature neurons. To determine functional properties of newly generated neurons, we administered NMDA to the lateral ventricle. As an in vivo response, we assessed extracellular-regulating kinase (ERK) phosphorylation. The newly generated neurons showed ERK phosphorylation by NMDA administration as seen in surrounding mature neurons. The number of newly generated neurons, which responded to NMDA receptor stimulation, increased with time after MK-801 treatment. The present study provides evidence that newly generated neurons in the adult hippocampus after NMDA receptor blockade acquire biochemical function in vivo.

Animals↗

PubMed2004

Characterization of BrdU-positive neurons induced by transient global ischemia in adult hippocampus.

Neurogenesis in the brain continues throughout life and is promoted by brain insults including ischemia. There is no critical conclusion, however, about whether proliferated cells acquire neuronal function after ischemia. Transient global ischemia was produced by a four-vessel occlusion procedure in rats (n = 54). To label proliferative cells, rats were administrated with a single dose of 5-bromo-2'-deoxyuridine (BrdU) at 4, 6, 8, 10, 13, or 15 days after ischemia. Increases in BrdU-positive cells were detected in the hippocampal dentate gyrus at 5, 7, and 9 days after ischemia. To determine the phenotype of BrdU-positive cells, BrdU was administrated twice daily for 3 consecutive days during 6 to 8 days after ischemia. A basic helix-loop-helix transcription factor NeuroD at 7 and 14 days and an immature migrating neuronal marker doublecortin at 14 days after ischemia were expressed transiently in proliferative cells. These proliferative cells after ischemia differentiated to the phenotype of neuron at 28 days after ischemia. Furthermore, BrdU-positive neurons showed phosphorylation of extracellular signal-regulated kinase (ERK) by intracerebroventricular injection of N-methyl-D-aspartate (NMDA) at 28 and 56 days after ischemia as seen in surrounding mature neurons. The number of BrdU-positive neurons, which responded to NMDA stimulation, increased with time after ischemia and was greater than that of sham-operated animals. The present study provides evidence for in vivo ERK phosphorylation in response to NMDA stimulation of BrdU-positive neurons in the adult hippocampus after transient forebrain ischemia.

Animals↗

PubMed2004

Effects of monochlorobimane on cerebral ischemia-induced damage to mitochondria.

A possible involvement of inhibitory effects of monochlorobimane (MCB) on the opening of mitochondrial permeability transition (MPT) pore in the cerebroprotection against the ischemic brain injury was examined. MCB (1 mM) inhibited the opening of MPT pore in vitro. Sustained cerebral ischemia was induced by injecting 900 microspheres (48 microm in diameter) into the right hemisphere of rats. At 12 to 72 h after microsphere embolism (ME), the mitochondrial activity was determined histochemically by staining cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) of the brain sections. The COX and SDH stainings in the hippocampus were observed intensively in the pyramidal neurons in the CA2-3 and dentate gyrus rather than those in the CA-1 region. The staining was decreased with time after the embolism. Pretreatment with 10 microg/animal MCB 30 min prior to the embolism significantly attenuated the ME-induced reduction in the staining of COX and SDH in the hippocampus, but not in the pariatal cortex. The results suggest that prevention of the opening of MPT pore by MCB may play an important role in the cerebroprotection against cerebral ischemic injury.

Animals↗

PubMed2004

The effects of monobromobimane on calcium and phenylarsineoxide-induced mitochondrial swelling and cytochrome C release in isolated brain mitochondria.

A possible involvement of inhibitory effects of monobromobimane (MBM), a thiol reagent, on the swelling and the release of cytochrome c in the isolated brain mitochondria was examined. MBM dose-dependently inhibited the calcium and phenylarsineoxide-induced mitochondrial swelling and cytochrome c release. Significant relationships between mitochondrial swelling and cytochrome c release were detected. Furthermore, effects of in vivo treatment with MBM on neuronal cell damage after transient (15 min) global ischemia in rats were examined. Infusion of MBM (1 or 3 microg/animal) to cerebral ventricles attenuated an increased number of TUNEL-positive cells and neuronal cell death in the hippocampal CA1 region at 72 h of reperfusion. These results suggest that MBM may have an ability to inhibit mitochondria-associated apoptotic pathways through attenuation of the mitochondrial swelling and the release of cytochrome c.

Animals↗

PubMed2003

Rapid appearance of pathological changes of neurons and glia cells in the cerebellum of microsphere-embolized rats.

Neuropathological changes in the cerebellar cortex of microsphere-embolized rats were studied at 30 min and 3 h after the embolism. Necrotic processes including a sponge-like vacuolation in the molecular layer, a vague outline of some Purkinje cells, and a few pyknotic granule cells having small and dark profiles were identified at sometime between 30 min and 3 h after microsphere-induced embolism in Nissl staining. Glial fibrillary acidic protein staining shows an apparent reduction in the number of Bergmann glial processes in some of the areas where there was necrosis of the molecular layer and poor astroglia processes in the areas subjacent to the pyknotic granule cells. These data demonstrate that within a short time, microsphere-induced cerebral ischemia produces necrosis of cerebellar neurons (i.e. Purkinje and granule cells) and changes in cerebellar glia cells (i.e. Bergmann and astroglia cells), and that these neuropathological changes are secondary phenomenon caused by microsphere blockage of cerebellar blood flow.

Animals↗