Takuo Hashimoto

Anatomical and functional recovery by embryonic stem cell-derived neural tissue of a mouse model of brain damage

We have treated undifferentiated mouse embryonic stem (ES) cells with all-trans retinoic acid (RA) to induce differentiation in vitro into neuron-like cells with good cell viability for use as a graft. Furthermore, we asked whether the RA-induced neuron-like cells restored neurological dysfunction. To this end, the cells were transplanted into right hemiplegia model of mice, developed by a cryogenic injury of motor cortex. Motor function of the recipients was gradually improved, whereas little improvement was observed in control mice. The lesion showed clustering of mature and almost mature neuron-like cells in mice transplanted with the RA-treated cells. The grafted cells had synaptic vesicles. This finding may suggest their maturation and synaptic connection in the recipient brain. Even though further study is necessary to elucidate molecular and cellular mechanisms responsible for the functional recovery, we consider that the ES cells may have advantage for use as a donor source in various neurological disorders including motor dysfunction.

Noggin and basic FGF were implicated in forebrain fate and caudal fate, respectively, of the neural tube-like structures emerging in mouse ES cell culture

We developed neural tube-like structures accompanying neural crest-like cells by treating embryonic stem (ES) cells with retinoic acid. The structures contained pseudostratified Nestin+Vimentin+ neuroepithelial cells surrounded by Masson staining+ basement membrane. βIIItubulin+Synaptophysin+ mature neurons and glial fibrillary acidic protein (GFAP)+ glial cells dispersed outside of the membrane. Addition of Noggin to the culture induced prominent proliferation of the neuroepithelial cells, leading to epithelial hyperstratification of the structures. mRNAs of transcription factors essential for forebrain development such as Emx1/2 and Pax6 were specifically expressed and Islet1+Lim1/2- motoneurons appeared by the addition of Noggin. In contrast, basic fibroblast growth factor (bFGF) promoted enlargement of central lumen and elongation of the structures. mRNAs of caudal markers, Gbx2, Cdx2 and Hoxb4/9 were expressed and Lim1/2+ spinal motoneurons appeared by the addition of bFGF. Addition of BMP-4 similarly brought about mild enlargement of central lumen of the structures. Interestingly, the addition of BMP-4 induced Slug+ neural crest-like cells surrounding the tube-like structures. mRNAs of Snail and dHand, other markers for neural crest cells, were also expressed by the addition of BMP-4. These results suggest that Noggin lead the neural-tube like structures to forebrain fate, whereas bFGF was involved in the caudalization. BMP-4 was implicated in emergence of the neural crest-like cells. Differentiation of ES cells by the present methods may mimic neurulation and subsequent neural development of early embryos, and elucidates the opposite effects of Noggin and bFGF for the neural tube development.

Transplantation of motoneurons derived from MASH1-transfected mouse ES cells reconstitutes neural networks and improves motor function in hemiplegic mice.

Mouse embryonic stem (ES) cells were transfected with a MASH1 expression vector and G418-resistant cells were selected. The MASH1-transfected cells became neuron-like appearance and expressed betaIIItubulin and panNCAM. Glial fibrillary acidic protein (GFAP) and galactocerebroside (GalC)-expressing cells were rarely detected. Half of the neural cells differentiated into the Islet1+ motoneuron lineage. Thus, we obtained motoneuron lineage-enriched neuronal cells by transfection of ES cells with MASH1. A hemiplegic model of mice was developed by cryogenic injury of the motor cortex, and motoneuron lineage-enriched neuronal cells were transplanted underneath the injured motor cortex neighboring the periventricular region. The motor function of the recipients was assessed by a beam walking and rotarod tests, whereby the results gradually improved, but little improvement was observed in vehicle injected control mice. We found that the grafted cells not only remained close to the implantation site, but also exhibited substantial migration, penetrating into the damaged lesion in a directed manner up to the cortical region. Grafted neuronal cells that had migrated into the cortex were elongated axon-positive for neurofilament middle chain (NFM). Synaptophysin immunostaining showed a positive staining pattern around the graft, suggesting that the transplanted neurons interacted with the recipient neurons to form a neural network. Our study suggests that the motoneuron lineage can be induced from ES cells, and grafted cells adapt to the host environment and can reconstitute a neural network to improve motor function of a paralyzed limb.

Recent Advances in Neurotraumatology

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Novel effects of edaravone on human brain microvascular endothelial cells revealed by a proteomic approach

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a free radical scavenger used for acute ischemic stroke. However, it is not known whether edaravone works only as a free radical scavenger or possess other pharmacological actions. Therefore, we elucidated the effects of edaravone on human brain microvascular endothelial cells (HBMECs) by 2 dimensional fluorescence difference gel electrophoresis (2D-DIGE). We found 38 protein spots the intensity of which was significantly altered 1.3 fold on average (p< 0.05) by the edaravone treatment and successfully identified 17 proteins of those. Four of those 17 proteins were cytoskeleton proteins or cytoskeleton-regulating proteins. Therefore, we subsequently investigated the change of size and shape of the cells, the actin network, and the tight junction of HBMEC by immunocytochemistry. As a result, most edaravone-treated HBMECs became larger and rounder compared with those that were not treated. Furthermore, edaravone-treated HBMECs formed gathering zona occludens (ZO)-1, a tight junction protein, along the junction of the cells. In addition, we found that edaravone suppressed interleukin (IL)-1β-induced secretion of monocyte chemoattractant protein-1 (MCP-1), which was reported to increase cell permeability. We found a novel function of edaravone is the promotion of tight junction formations of vascular endothelial cells partly via the down-regulation of MCP-1 secretion. These data provide fundamental and useful information in the clinical use of edaravone in patients with cerebral vascular diseases.

Increased distribution of carboplatin, an anti-cancer agent, to rat brains with the aid of hyperbaric oxygenation.

1. The distribution of an anti-cancer agent carboplatin to brains was investigated in combination with hyperbaric oxygenation treatment in rats. 2. After intravenous administration of carboplatin (30 mg kg−1) to male Wistar rats, elimination curves of plasma drug concentrations plotted against a time of 45 min were not different with or without hyperbaric oxygenation (at 0.20–0.25 MPa for last 20 min) treatments. 3. Carboplatin concentrations of livers, lungs and kidneys in each group were similar at the endpoint of hyperbaric oxygenation treatment. 4. Under these atmosphere conditions (at 0.10 MPa), carboplatin concentration was at an undetectable level in rat brains (<0.1 µg g−1 tissue, n = 6). On the contrary, carboplatin was detected in all brains tested at the levels of 0.5 ± 0.3 µg g−1 tissue (mean and standard deviation (SD), n = 6), 0.8 ± 0.5 µg g−1 tissue, and 0.4 ± 0.2 µg g−1 tissue in combination with hyperbaric oxygenation at 0.20, 0.22, and 0.25 MPa, respectively, at the endpoint of hyperbaric oxygenation treatment. 5. The results suggest that carboplatin could be uptaken into rat brains at the detectable levels by the aid of hyperbaric oxygenation, consistently with the reported findings of enhanced transendothelial permeability and improved clinical efficacy of carboplatin combined hyperbaric oxygenation therapy.

Dissecting aneurysm of the vertebro-basilar system. Surgical treatment in cases with brain stem ischemia.

椎骨動脈脳底動脈系の解離性動脈瘤は比較的稀な疾患と考えられてきたが, 近年, 本疾患の可能性を考慮し, DSAなどの神経放射線学的診断が積極的に行われ, 少なからず経験されるようになった.しかし臨床診断治療は困難なことが多く, mortalityの高い疾患で, 治療法については確立されておらずまだ論議の多いところである.筆者らは脳幹部の虚血症状で発症し, 外側延髄症候群を呈した椎骨脳底動脈系の解離性脳動脈瘤4例を経験したので, 症状, 血管撮影所見, 特に虚血症状で発生した症例に対する外科的治療法の選択について報告した.解離性動脈瘤が椎骨動脈に留まっている間に時期を失することなく積極的に外科的治療を行う必要がある.血行再建術を施行し, 解離性動脈瘤が消失した1例を経験した.術後血行動態の変化が生じ, 解離性動脈瘤のorificeが閉鎖されることも期待でき, 椎骨動脈のclippingだけで.なく, 血行再建術も考慮すべきである.

Analysis of intracranial pressure pulse waveform and brain capillary morphology in type 2 diabetes mellitus rats.

Diabetes mellitus in neurosurgical patients is known to be a disease with high risks and severe outcomes. However, the mechanism by which diabetes mellitus induces dysfunction of brain tissue is not well known. The hypothesis of this study was that the damage to brain microvasculature in diabetes mellitus results in impaired compliance of the brain. Pathological changes associated with type II diabetes were investigated using a rat model. Pathophysiological changes in diabetic brain tissue were also investigated to confirm cerebral compliance by analyzing intracranial pressure waveforms. Pathologic findings revealed thickening of the basement membrane and fibrous collagen infiltration into the inner basement membrane of the brain microvasculature in diabetes mellitus. Analysis of intracranial pressure waveforms revealed that the P2 portion increased in diabetic rats compared to the control and was increased further with the increase in intracranial pressure. Analysis of the differential pressure curve, with respect to time, demonstrated that intracranial elasticity showed a concomitant increase. Pathologic findings and intracranial pressure waveforms were consistent with changes in brain microvasculature in diabetes mellitus. The increase of elasticity of brain tissue in diabetes mellitus may exacerbate the damage of intracranial disease.

Neuroimaging characteristics and growth pattern on magnetic resonance imaging in a 52-year-old man presenting with pituicytoma: a case report

IntroductionPituicytoma is a rare neoplasm of the neurohypophysis. To the best of our knowledge there have been no reports of pituicytoma in which long-term magnetic resonance imaging observation was performed. We calculated the doubling time of the tumor volume and described the growth pattern of a pituicytoma.Case presentationA 52-year-old Japanese man with a history of decreased libido was found to have a sellar and suprasellar mass. He underwent transsphenoidal surgery, but only a small specimen was obtained because of intraoperative bleeding. The tentative histological diagnosis was schwannoma. He noticed bitemporal hemianopsia 7 years later. A follow-up magnetic resonance imaging disclosed a tumor volume doubling time of 3830 days. Transcranial gross-total tumor resection was performed. The lesion consisted of elongated and plump tumor cells that were arranged in a fascicular or storiform pattern and were positive for S-100 protein and focally positive for glial fibrillary acidic protein. The final histological diagnosis was pituicytoma.ConclusionPituicytoma is a slow-growing tumor, but the growth rate may change during follow-up.

Intracellular lipid content of liver and skeletal muscle in patients with adult growth hormone deficiency without diabetes mellitus

SUMMARY BACKGROUND Insulin resistance (IR) and visceral obesity are often observed in adult growth hormone deficiency patients (AGHDs). However, there is little information regarding the intrahepatic lipid (IHL) or the intramyocellular lipid (IMCL) content and their association with IR in AGHDs. The aim of this study was to directly assess IHL and IMCL in AGHDs by proton magnetic resonance spectroscopy and to evaluate the association of lipid levels with IR. METHODS Appropriate hormone replacement therapy (RT) other than GH and estrogen was prescribed before evaluation. Ten AGHDs (aged 23-75 years) without diabetes or elevation of aminotransferases were examined the percent body fat, visceral fat area (VFA), IHL, IMCL, adipokines and glucose metabolism. In two AGHDs, changes of these parameters were evaluated after GHRT. RESULTS Visceral obesity and metabolic syndrome was found in 100% and in 80% of the patients, respectively. IHL was significantly higher than that in non-obese healthy controls (12.5 ± 4.6 vs. 0.69 ± 0.46%, M ± SE, p = 0.0330), while IMCL did not differ between AGHDs and controls (528.8 ± 137.2 vs. 378 ± 51.1 mM, p = 0.2728). Homeostasis model assessment of IR was significantly correlated with IHL (r = 0.896, p = 0.0001) and IMCL (r = 0.749, p = 0.0102), but not with the VFA or percent truncal fat mass. A decrease of IHL and improvement of glucose tolerance were observed in the two patients after 6 M GHRT. CONCLUSION These results demonstrated that IHL, but not IMCL, may increase in AGHDs, and that IHL may associate with IR. GHRT may decrease IHL along with amelioration of IR.