Faculty Profiles - YAMASHITA Toru

Papers - YAMASHITA Toru

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Therapeutic approaches to vascular protection in ischemic stroke. Reviewed

Toru Yamashita, Koji Abe

Acta medica Okayama 65 ( 4 ) 219 - 23 2011.8

Language：English Publishing type：Research paper (scientific journal)

Reperfusion with recombinant tissue plasminogen activator (tPA) sometimes causes catastrophic hemorrhagic transformation (HT) in the ischemic brain. Consequently, the application of tPA has been strictly limited. Recent studies have indicated that matrix metalloproteinases (MMPs), especially MMP-9, play a critical role in blood brain barrier (BBB) disruption in the ischemic brain, leading to brain edema and HT. In the ischemic brain, free radicals and exogenous tPA itself can trigger MMP-9 activation through several signaling pathways containing LDL receptor-related protein (LRP) and proteinase-activated receptor 1 (PAR1). Therapeutic targeting of free radicals and MMP-9/t-PA related signaling pathways might be promising approaches to minimizing catastrophic HT in acute stroke patients. We provide an overview of the available scientific reports to improve our understanding of the mechanisms leading to HT, and highlight recent progress in the development of new therapeutic strategies for preventing HT in the post-stroke brain.

Neurological and pathological improvements of cerebral infarction in mice with platinum nanoparticles. Reviewed International journal

Motonori Takamiya, Yusei Miyamoto, Toru Yamashita, Kentaro Deguchi, Yasuyuki Ohta, Yoshio Ikeda, Tohru Matsuura, Koji Abe

Journal of neuroscience research 89 ( 7 ) 1125 - 33 2011.7

Language：English Publishing type：Research paper (scientific journal)

Ischemic stroke is a major, urgent neurologic disorder in which reactive oxygen species (ROS) are deeply involved in the detrimental effects. Platinum nanoparticle (nPt) species are a novel and strong scavenger of such ROS, so we examined the clinical and neuroprotective effects of nPts in mouse ischemic brain. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min. Upon reperfusion, nPt or vehicle was administered intravenously. At 48 hr after the tMCAO, motor function, infarct volume, immunohistochemistry of neurovascular components (endothelial NAGO, tight junctional occludin, and basal laminal collagen IV), and zymography for MMP-9 activity were examined. Superoxide anion generation at 2 hr after tMCAO was determined with oxidized hydroethidine. Compared with vehicle, treatment with nPts significantly improved the motor function and greatly reduced the infarct volume, especially in the cerebral cortex. Immunohistochemical analyses revealed that tMCAO resulted in a minimal decrease of NAGO and occludin but a great decrease of collagen IV and a remarkable increase of MMP-9. Treatment with nPts greatly reduced this decrease of collagen IV and activation of MMP-9 and, with large reductions of MMP-9 activation on zymography and superoxide production. The present study demonstrates that treatment with nPts ameliorates the neurological scores with a large reduction in infarct size as well as the preservation of outer components of the neurovascular unit (collagen IV) and inactivation of MMP-9. A strong reduction of superoxide anion production by nPts could account for such remarkable neurobehavioral and neuroprotective effects on ischemic stroke.

DOI： 10.1002/jnr.22622

Comparisons of acoustic function in SCA31 and other forms of ataxias. Reviewed International journal

Yoshio Ikeda, Makiko Nagai, Tomoko Kurata, Toru Yamashita, Yasuyuki Ohta, Shoko Nagotani, Kentaro Deguchi, Yasushi Takehisa, Yoshihiko Shiro, Tohru Matsuura, Koji Abe

Neurological research 33 ( 4 ) 427 - 32 2011.5

Language：English Publishing type：Research paper (scientific journal)

OBJECTIVE: To investigate whether acoustic impairment can be one of the characteristic extracerebellar symptoms in sporadic and hereditary ataxias including spinocerebellar ataxia type 31 (SCA31). METHODS: We investigated genotypes of dominant ataxia families, and determined a frequency of each form in our cohort of 154 families. Acoustic function in the groups of various forms of ataxia with multiple system atrophy of cerebellar predominance (MSA-C), cortical cerebellar atrophy (CCA), and hereditary ataxias including SCA31 was evaluated by using audiogram and brainstem auditory evoked potentials (BAEPs). RESULTS: Genetic analysis of dominant ataxia families revealed that a frequency of SCA31 in our cohort was fewer than that reported from other areas of Japan, indicating that SCA31 is not widely distributed throughout Japan. Results of audiogram showed no significant difference of hearing levels among ataxic groups, and those of BAEPs did not support inner ear dysfunction in SCA31 in which hearing loss had initially been suggested as one of its characteristic symptoms. CONCLUSION: This study suggests that acoustic impairment is neither specific to SCA31, MSA-C and CCA nor useful in making a differential diagnosis among them.

DOI： 10.1179/1743132810Y.0000000011

Protection against ischemic stroke damage by synergistic treatment with amlodipine plus atorvastatin in Zucker metabolic rat. Reviewed International journal

Hiromi Kawai, Shoko Deguchi, Kentaro Deguchi, Toru Yamashita, Yasuyuki Ohta, Yoshio Omote, Tomoko Kurata, Yoshio Ikeda, Tohru Matsuura, Koji Abe

Brain research 1382 308 - 14 2011.3

Language：English Publishing type：Research paper (scientific journal)

Ischemic stroke is a major neurologic disorder and a leading cause of disability and death in the world. We compared neuroprotective effects of single or combination therapy of amlodipine (AM) and atorvastatin (AT) in such a metabolic syndrome model Zucker rat. The animals were pretreated with vehicle, AM, AT, or the combination of AM plus AT for 28days, and physical and serum parameters were analyzed, then 90min of transient middle cerebral artery occlusion (tMCAO), was performed followed by immunohistochemical analyses at 24h. Without affecting serum levels of lipids, adiponectin, and leptin, the combination therapy of AM plus AT ameliorated the post-ischemic brain weight increase. The single treatment with AM or AT itself exerted neuroprotective effects with reducing inductions of MMP-9 and AT2R, as well as with preserving collagen IV, and the combination therapy of AM plus AT showed a further synergistic benefit against acute ischemic neural damages. Single AT was more protective on these 3 molecules than single AM at this time point of 24h after tMCAO. Thus, the combination therapy with AM plus AT extended the neuroprotectives effect of single treatment with AM or AT on a part of neurovascular unit and a hypertension-related receptor.

DOI： 10.1016/j.brainres.2011.01.062

Synergistic benefit of combined amlodipine plus atorvastatin on neuronal damage after stroke in Zucker metabolic rat. Reviewed International journal

Hiromi Kawai, Shoko Deguchi, Kentaro Deguchi, Toru Yamashita, Yasuyuki Ohta, Jingwei Shang, Fengfeng Tian, Xuemei Zhang, Ning Liu, Wentao Liu, Yoshio Ikeda, Tohru Matsuura, Koji Abe

Brain research 1368 317 - 23 2011.1

Language：English Publishing type：Research paper (scientific journal)

Stroke is a major neurologic disorder and a leading cause of death in the world. We compared neuroprotective effects of single or combination therapy of amlodipine (AM) and atorvastatin (AT) in such a metabolic syndrome model Zucker rat after 90 min of transient middle cerebral artery occlusion (tMCAO). The animals were pretreated with vehicle, AM, AT, or the combination of AM plus AT for 28 days, and at 24h of tMCAO, infarct volume and immunohistochemical analyses were performed. The combination of AM plus AT treatment decreased the infarct volume stronger than each single treatment with AM or AT. The numbers of positive cells of oxidative stress markers such as 8-hydroxy-2'-deoxyguanosin (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), and advanced end glycation products (AGE) and inflammation markers such as tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1(MCP-1) decreased dramatically in the combination-treated group compared with single AM- or AT-treated group. The present study showed that single AM or AT treatment showed neuroprotective effects both with antioxidative and anti-inflammatory mechanisms, but combination therapy of AM plus AT presented a further synergistic benefit in acute ischemic neural damages.

DOI： 10.1016/j.brainres.2010.10.046

Strong neurogenesis, angiogenesis, synaptogenesis, and antifibrosis of hepatocyte growth factor in rats brain after transient middle cerebral artery occlusion. Reviewed International journal

Jingwei Shang, Kentaro Deguchi, Yasuyuki Ohta, Ning Liu, Xuemei Zhang, Fengfeng Tian, Toru Yamashita, Yoshio Ikeda, Tohru Matsuura, Hiroshi Funakoshi, Toshikazu Nakamura, Koji Abe

Journal of neuroscience research 89 ( 1 ) 86 - 95 2011.1

Language：English Publishing type：Research paper (scientific journal)

Hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor (GDNF) are strong neurotrophic factors. However, their potentials in neurogenesis, angiogenesis, synaptogenesis, and antifibrosis have not been compared. Therefore, we investigated these effects of HGF and GDNF in cerebral ischemia in the rat. Wistar rats were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO). Immediately after reperfusion, HGF or GDNF was given by topical application. BrdU was injected intraperitoneally twice daily 1, 2, and 3 days after tMCAO. On 14 day, we histologically evaluated infarct volume, antiapoptotic effect, neurogenesis, angiogenesis, synaptogenesis, and antifibrosis. Both HGF and GDNF significantly reduced infarct size and the number of TUNEL-positive cells, but only HGF significantly increased the number of BrdU-positive cells in the subventricular zone, and 5'-bromo-2'-deoxyuridine -positive cells differentiated into mature neurons on the ischemic side. Enhancement of angiogenesis and synaptogenesis at the ischemic boundary zone was also observed only in HGF-treated rats. HGF significantly decreased the glial scar formation and scar thickness of the brain pia mater after tMCAO, but GDNF did not. Our study shows that both HGF and GDNF had significant neurotrophic effects, but only HGF can promote the neurogenesis, angiogenesis, and synaptogenesis and inhibit fibrotic change in brains after tMCAO.

DOI： 10.1002/jnr.22524

Vascular protection and restorative therapy in ischemic stroke. Reviewed International journal

Toru Yamashita, Kentaro Deguchi, Shoko Nagotani, Koji Abe

Cell transplantation 20 ( 1 ) 95 - 7 2011

Language：English Publishing type：Research paper (scientific journal)

Possible strategies for treating stroke include: 1) thrombolytic therapy with tissue plasminogen activator (tPA): restoring cerebral blood flow in the acute phase of ischemic stroke but sometimes causing hemorrhagic transformation (HT); 2) stem cell therapy: the repair of disrupted neuronal networks with newly born neurons in the chronic phase of ischemic stroke. Firstly, we estimated the vascular protective effect of a free radical scavenger, edaravone, in the tPA-treated rat model of middle cerebral artery occlusion. Edaravone prevented dramatically decreased the hemorrhagic transformation and improved the neurologic score and survival rate of tPA-treated rats. Secondly, we attempted to restore brain tissue using a novel biomaterial, polydimethysiloxane-tetraethoxysilane (PDMS-TEOS) hybrid with or without vascular endothelial growth factor (VEGF), and we could show that implantation of a PDMS-TEOS scaffold with VEGF might be effective for treating old brain infarction or trauma. In the future, we will combine these strategies to develop more effective therapies for treatment of strokes.

DOI： 10.3727/096368910X532800

Advances in induced Pluripotent Stem cell therapy for neurological diseases Reviewed

Toru Yamashita, Hiromi Kawai, Koji Abe

Japanese Journal of Neurosurgery 20 ( 8 ) 585 - 588 2011

Language：English Publishing type：Research paper (scientific journal) Publisher：Japanese Congress of Neurological Surgeons

Induced Pluripotent Stem (iPS) cells are regarded to be potential supply source of cells for application to cell replacement therapies. Recent scientific papers indicated that human iPS cells are capable of differentiating into glutamatergic neurons, dopaminergic neurons, and motor neurons. However, tumorigenesis in iPS cells is a serious problem which should be overcome for clinical application. Recently, we evaluated the influence of ischemic condition on undifferentiated iPS cells within a transient Middle Cerebral Artery Occlusion (MCAO) using a mouse model. Undifferentiated iPS cells (5 × 105) were injected into the ipsilateral striatum and cortex, which were considered as the ischemic boundary zone at 24 hours after MCAO. Ischemic brains treated with iPS cells tended to form teratoma with larger volume compared with those in intact brains. c~Myc, Oct3/4 and Sox2 were strongly expressed in iPS-derived tumors of the ischemic brain. Above results indicated that the transcriptional factors integrated into the genome by retrovirus vectors might promote teratoma formation in the ischemic brain. Potential interaction between integrated transcriptional factors and iPS cell characteristics needs to be kept in mind, for the development of transplantation therapy.

DOI： 10.7887/jcns.20.585

Tumorigenic development of induced pluripotent stem cells in ischemic mouse brain. Reviewed International journal

Toru Yamashita, Hiromi Kawai, Fengfeng Tian, Yasuyuki Ohta, Koji Abe

Cell transplantation 20 ( 6 ) 883 - 91 2011

Language：English Publishing type：Research paper (scientific journal)

Induced pluripotent stem (iPS) cells may provide cures for various neurological diseases. However, undifferentiated iPS cells have high tumorigenicity, and evaluation of the cells fates, especially in pathologic condition model, is needed. In this study, we demonstrated the effect of ischemic condition to undifferentiated iPS cells fates in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells were characterized with immunofluorescent staining. The iPS cells (5 × 10⁵) were injected into ipsilateral striatum and cortex after 24 h of MCAO. Histological analysis was performed from 3 to 28 days after cell transplantation. iPS cells in ischemic brain formed teratoma with higher probability (p < 0.05) and larger volume (p < 0.01) compared with those in intact brain. Among the four transcriptional factors to produce iPS cells, c-Myc, Oct3/4, and Sox2 strongly expressed in iPS-derived tumors in ischemic brain (p < 0.01). Additionally, expression of matrix metalloproteinase-9 (MMP-9) and phosphorylated vascular endothelial growth factor receptor2 (phospho-VEGFR2) were significantly increased in iPS-derived tumors in the ischemic brain (p < 0.05). These results suggest that the transcriptional factors might increase expression of MMP-9 and activate VEGFR2, promoting teratoma formation in the ischemic brain. We strongly propose that the safety of iPS cells should be evaluated not only in normal condition, but also in a pathologic, disease model.

DOI： 10.3727/096368910X539092

In vivo optical imaging of early-stage apoptosis in mouse brain after transient cerebral ischemia. Reviewed International journal

Ning Liu, Kentaro Deguchi, Jingwei Shang, Xuemei Zhang, Fengfeng Tian, Toru Yamashita, Yasuyuki Ohta, Yoshio Ikeda, Tohru Matsuura, Koji Abe

Journal of neuroscience research 88 ( 16 ) 3488 - 97 2010.12

Language：English Publishing type：Research paper (scientific journal)

Apoptosis is one of the mechanisms contributing to neuronal degeneration in ischemic stroke. In vivo imaging of annexin V (A5) was performed at 12 hr, 24 hr, 48 hr, and 4 days after 90-min transient middle cerebral artery occlusion (tMCAO) in mice with a fluorescent protein Cy5.5. Immunohistochemistry for heat shock protein 70 (HSP70), A5, and TUNEL were also performed with brain sections after the tMCAO. In vivo fluorescence was strongly observed at 48 hr over the head, especially with removal of both head skin and skull bone. Zonal ex vivo fluorescent signals were surrounding the ischemic core, and double-positive cells with Cy5.5/exogenous A5 antibody were found in this area. HSP70 was observed at the peak time of 24 hr; A5 became detectable at 12 hr, with increasing numbers until 48 hr. The number of TUNEL-positive cells increased at 24 hr and retained the high level until 4 days, showing a dissociating temporal pattern with A5. Double-positive cells for A5/TUNEL reached their peak at 48 hr. All the data suggest that some cells still have a chance to be rescued for a long period after acute cerebral ischemia. The in vivo Cy5.5 fluorescence representing A5 signal spatially surrounding the ischemic core temporally detects an early-stage apoptosis after cerebral ischemia.

DOI： 10.1002/jnr.22489

Free radical scavenger edaravone administration protects against tissue plasminogen activator induced oxidative stress and blood brain barrier damage. Reviewed International journal

Violeta Lukic-Panin, Kentaro Deguchi, Toru Yamashita, Jingwei Shang, Xuemei Zhang, FengFeng Tian, Ning Liu, Hiromi Kawai, Tohru Matsuura, Koji Abe

Current neurovascular research 7 ( 4 ) 319 - 29 2010.11

Language：English Publishing type：Research paper (scientific journal)

One of the therapeutics for acute cerebral ischemia is tissue plasminogen activator (t-PA). Using t-PA after 3 hour time window increases the chances of hemorrhage, involving multiple mechanisms. In order to show possible mechanisms of t-PA toxicity and the effect of the free radical scavenger edaravone, we administered vehicle, plasmin, and t-PA into intact rat cortex, and edaravone intravenously. Plasmin and t-PA damaged rat brain with the most prominent injury in t-PA group on 4-HNE, HEL, and 8-OHdG immunostainings. Such brain damage was strongly decreased in t-PA plus edaravone group. For the neurovascular unit immunostainings, occludin and collagen IV expression was decreased in single plasmin or t-PA group, which was recovered in t-PA plus edaravone group. In contrast, matrix metalloproteinase-9 intensity was the strongest in t-PA group, less in plasmin, and was the least prominent in t-PA plus edaravone group. In vitro data showed a strong damage to tight junctions for occludin and claudin 5 in both administration groups, while there were no changes for endothelial (NAGO) and perivascular (GFAP) stainings. Such damage to tight junctions was recovered in t-PA plus edaravone group with similar recovery in Sodium-Fluorescein permeability assay. Administration of t-PA caused oxidative stress damage to lipids, proteins and DNA, and led to disruption of outer parts of neurovascular unit, greater than the effect in plasmin administration. Additive edaravone ameliorated such an oxidative damage by t-PA with protecting outer layers of blood-brain barrier (in vivo) and tight junctions (in vitro).

In vivo imaging of autophagy in a mouse stroke model. Reviewed International journal

FengFeng Tian, Kentaro Deguchi, Toru Yamashita, Yasuyuki Ohta, Nobutoshi Morimoto, Jingwei Shang, Xuemei Zhang, Ning Liu, Yoshio Ikeda, Tohru Matsuura, Koji Abe

Autophagy 6 ( 8 ) 1107 - 14 2010.11

Language：English Publishing type：Research paper (scientific journal)

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the periischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.

Antiapoptotic and antiautophagic effects of glial cell line-derived neurotrophic factor and hepatocyte growth factor after transient middle cerebral artery occlusion in rats. Reviewed International journal

Jingwei Shang, Kentaro Deguchi, Toru Yamashita, Yasuyuki Ohta, Hanzhe Zhang, Nobutoshi Morimoto, Ning Liu, Xuemei Zhang, Fengfeng Tian, Tohru Matsuura, Hiroshi Funakoshi, Toshikazu Nakamura, Koji Abe

Journal of neuroscience research 88 ( 10 ) 2197 - 206 2010.8

Language：English Publishing type：Research paper (scientific journal)

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser(2448) (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to beta-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways.

DOI： 10.1002/jnr.22373

Tridermal tumorigenesis of induced pluripotent stem cells transplanted in ischemic brain. Reviewed International journal

Hiromi Kawai, Toru Yamashita, Yasuyuki Ohta, Kentaro Deguchi, Shoko Nagotani, Xuemei Zhang, Yoshio Ikeda, Tohru Matsuura, Koji Abe

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 30 ( 8 ) 1487 - 93 2010.8

Language：English Publishing type：Research paper (scientific journal)

Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5 x 10(5)) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled.

DOI： 10.1038/jcbfm.2010.32

Temporal and spatial differences of multiple protein expression in the ischemic penumbra after transient MCAO in rats. Reviewed International journal

Xuemei Zhang, Kentaro Deguchi, Toru Yamashita, Yasuyuki Ohta, Jingwei Shang, Fengfeng Tian, Ning Liu, Violeta Lukic Panin, Yoshio Ikeda, Tohru Matsuura, Koji Abe

Brain research 1343 143 - 52 2010.7

Language：English Publishing type：Research paper (scientific journal)

Temporal and spatial differences and relationships of proteins relating to the ischemic penumbra were examined at 1, 3, 12, 24, and 48 h after 90 min of transient middle cerebral artery occlusion (tMCAO) in rats. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the apparent infarction focus first appeared at 1h after tMCAO, which then largely matured at 24h. Immunohistochemistry and Western blot indicated no or trace levels of c-fos, hypoxia inducible factor-1 alpha (HIF-1 alpha), heat shock protein 70 (HSP70), and annexin V (A5) positive cells in the sham control brain. Expression of c-fos increased quickly and widely within and outside of the affected arterial territory (peak at 1h), and that of HIF-1 alpha reached the maximum at 12h in a smaller area than c-fos. HSP70 began to be induced during the first few hours after tMCAO, peaked at 24h, then decreased within 48 h, while A5 was slightly expressed at 3h, then gradually increased until 48 h. Double immunofluorescent analyses showed that the colocalization rates of c-fos/HIF-1 alpha, HIF-1 alpha/HSP70, HSP70/A5, and A5/TUNEL were 40.6%, 58.4%, 42.1% and 61.0%, respectively. These data suggest that multiple molecular penumbra exist after 90 min of tMCAO in the rat brain where several different proteins participate in different temporal and spatial expression patterns. Thus, there is a window for rescue of ischemic neural cells from 12 to 48 h after injury.

DOI： 10.1016/j.brainres.2010.04.027

Dissociation and protection of the neurovascular unit after thrombolysis and reperfusion in ischemic rat brain. Reviewed International journal

Toru Yamashita, Tatsushi Kamiya, Kentaro Deguchi, Toshiki Inaba, Hanzhe Zhang, Jingwei Shang, Kazunori Miyazaki, Aiji Ohtsuka, Yasuo Katayama, Koji Abe

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 29 ( 4 ) 715 - 25 2009.4

Language：English Publishing type：Research paper (scientific journal)

In the ischemic brain, reperfusion with tissue plasminogen activator (tPA) sometimes causes catastrophic hemorrhagic transformation (HT); however, the mechanism remains elusive. Here, we show that the basement membrane, and not the endothelial cells, is vulnerable to ischemic/reperfusion injury with tPA treatment. We treated a spontaneously hypertensive rat model of middle cerebral artery occlusion (MCAO) with vehicle alone, tPA alone, or a free radical scavenger, edaravone, plus tPA. Light and electron microscopic analyses of each microvascular component revealed that the basement membrane disintegrated and became detached from the astrocyte endfeet in tPA-treated animals that showed HT. On the other hand, edaravone prevented the dissociation of the neurovascular unit, dramatically decreased the HT, and improved the neurologic score and survival rate of the tPA-treated rats. These results suggest that the basement membrane that underlies the endothelial cells is a key structure for maintaining the integrity of the neurovascular unit, and a free-radical scavenger can be a viable agent for inhibiting tPA-induced HT.

DOI： 10.1038/jcbfm.2008.164

Therapeutic strategy for ischemic stroke. Reviewed International journal

Toru Yamashita, Kentaro Deguchi, Yoshihide Sehara, Violeta Lukic-Panin, Hanzhe Zhang, Tatsushi Kamiya, Koji Abe

Neurochemical research 34 ( 4 ) 707 - 10 2009.4

Language：English Publishing type：Research paper (scientific journal)

Possible strategies for treating ischemic stroke include: (1) Neuroprotection: preventing damaged neurons from undergoing apoptosis in the acute phase of cerebral ischemia; (2) Stem cell therapy: the repair of broken neuronal networks with newly born neurons in the chronic phase of cerebral ischemia. Firstly, we studied the neuroprotective effect of a calcium channel blocker, azelnidipine, or a by-product of heme degradation, biliverdin, in the ischemic brain. These results revealed both azelnidipine and biliverdin had a neuroprotective effect in the ischemic brain through their anti-oxidative property. Secondly, we investigated the role of granulocyte colony-stimulating factor (G-CSF) by administering G-CSF to rats after cerebral ischemia and found G-CSF plays a critical role in neuroprotection. Lastly, we developed a restorative stroke therapy with a bio-affinitive scaffold, which is able to provide an appropriate environment for newly born neurons. In the future, we will combine these strategies to develop more effective therapies for treatment of strokes.

DOI： 10.1007/s11064-008-9842-2

Gene and stem cell therapy in ischemic stroke. Reviewed International journal

Toru Yamashita, Kentaro Deguchi, Shoko Nagotani, Tatsushi Kamiya, Koji Abe

Cell transplantation 18 ( 9 ) 999 - 1002 2009

Language：English Publishing type：Research paper (scientific journal)

Possible strategies for treating ischemic stroke include neuroprotection (preventing injured neurons from undergoing apoptosis in the acute phase of cerebral ischemia) and stem cell therapy (the repair of disrupted neuronal networks with newly born neurons in the chronic phase of cerebral ischemia). First, we estimated the neuroprotective effect of glial cell line-derived neurotrophic factor (GDNF) by administration of GFNF protein. GDNF protein showed a direct protective effect against ischemic brain damage. Pretreatment of animals with adenoviral vector containing GDNF gene (Ad-GDNF) 24 h before the subsequent transient middle cerebral artery occlusion (MCAO) effectively reduced infarcted volume. Secondly, we studied the neuroprotective effect of a calcium channel blocker, azelnidipine, or a by-product of heme degradation, biliverdin. Both azelnidipine and biliverdin had a neuroprotective effect in the ischemic brain through their antioxidative property. Lastly, we developed a restorative stroke therapy with a bioaffinitive scaffold, which is able to provide an appropriate platform for newly born neurons. In the future, we will combine these strategies to develop more effective therapies for treatment of strokes.

DOI： 10.3727/096368909X471233

Epigenetic regulation of neural cell differentiation plasticity in the adult mammalian brain. Reviewed International journal

Jun Kohyama, Takuro Kojima, Eriko Takatsuka, Toru Yamashita, Jun Namiki, Jenny Hsieh, Fred H Gage, Masakazu Namihira, Hideyuki Okano, Kazunobu Sawamoto, Kinichi Nakashima

Proceedings of the National Academy of Sciences of the United States of America 105 ( 46 ) 18012 - 7 2008.11

Language：English Publishing type：Research paper (scientific journal)

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

DOI： 10.1073/pnas.0808417105

Gelatin-siloxane hybrid scaffolds with vascular endothelial growth factor induces brain tissue regeneration. Reviewed International journal

Hanzhe Zhang, Tatsushi Kamiya, Takeshi Hayashi, Kanji Tsuru, Kentaro Deguchi, Violeta Lukic, Atsushi Tsuchiya, Toru Yamashita, Satoshi Hayakawa, Yoshio Ikeda, Akiyoshi Osaka, Koji Abe

Current neurovascular research 5 ( 2 ) 112 - 7 2008.5

Language：English Publishing type：Research paper (scientific journal)

In the brain after infarction or trauma, the tissue becomes pannecrotic and forms a cavity. In such situation, a scaffold is necessary to produce new tissue. In this study, we implanted a new porous gelatin-siloxane hybrid derived from gelatin and 3-(glycidoxypropyl) trimethoxysilane (gelatin-GPTMS) scaffolds into a brain defect, and investigated whether it makes a new brain tissue. In addition, vascular endothelial growth factor (VEGF) was added on gelatin-GPTMS scaffolds and its effect on tissue regeneration was examined. At 30 days after the implantation, the marginal territory of the scaffolds became occupied by newly formed tissue. Immunohistochemical analysis revealed that the new tissue was constituted by endothelial, astroglial and microglial cells, some of which were labeled for bromodeoxyuridine (BrdU). Addition of VEGF promoted numbers of these cells. Thus, combination of gelatin-GPTMS scaffolds and VEGF is preferable for brain regeneration.

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