2000 Fiscal Year Final Research Report Summary
The establishment of cell culture in hybrid type collagen and transplantation into intracerebral hemorrhage model rat brain
| Project/Area Number |
10671300
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cerebral neurosurgery
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| Research Institution | Kobe University |
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Principal Investigator |
KONDOH Takeshi Kobe Univ., School of Medicine, Dept. of Neurosurgery, Assistant Professor, 医学部, 助手 (50273769)
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| Co-Investigator(Kenkyū-buntansha) |
TAMAKI Norihiko Dept. of Neurosurgery, Professor, Chairman, 医学部, 教授 (10030941)
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| Project Period (FY) |
1998 – 2000
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| Keywords | Intracerebral hemorrhage / Neuronal Transplantation / Blood-brain Barrier / GFP / HUVEC / Electroporation |
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| Research Abstract |
With a purpose of ex vivo gene transfer into injured brain such as intracerebral hemorrhage, following three sets of preliminary experiments were performed.
(A) gene transfer by electroporation was attempted in the normal brain. The reporter gene pEGFP-Cl(25g /5 μl) was injected in the striatum of young adult rats and various ranges of square electrical impulses were applied by using a pair of electrodes that were placed in the striatum. After five days, histological examination revealed that the impulses of high voltage caused extensive tissue damage whereas impulses of lower range (200-400 mJ) resulted in the transfection of more than 300 cells per brain, which were widely distributed in the subependymal region of the lateral ventricle and extended long processes into the striatum.
(B) Human umbilical vein endothelial cells (HUVECs) were transplanted in athymic mouse brain and neovascularization of grafled endothelial cells was studied. HUVECs were transfected by a reporter gene pEGFPE
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-N1 in vitro and grafted stereotactically in unilateral striatum of adult nude mice. Histological studies in four weeks revealed that grafted HUVECs newly formed microvessels in brain, which were migrated and fused with host vessels. Intravenous injection of Evans Blue prior to sacrificing animals resulted m no extravasation of dye, indicating that a blood-brain barrier was formed by the grafted HUVECs. Immunohistochemistry demonstrated that host astrocytes extended glial feet on the grafted endothelial cells and a part of the newly formed vessels were positive with glucose transporter-1. These results indicate that endothelial cells from an ectopic origin have the potential to form a blood-brain barrier after grafting in the central nervous system.
(C) Neuronal progenitor cells have been widly studied with the purpose of regeneration of injured central nervous system. For the grafting of these cells, not pure single cell suspension of stem cells but spheres composed with progenitor cells have been reported to demonstrate improved survival after grafting. In this study, rat neuronal stem cells were obtained from E-14 rat subventriclur zone followed by free floating culture in EGF-containing medium as previously reported by Weiss et al. After four passage in four weeks, single stem cells were left to grow without dissociation for two months by changing the medium weekly. The spheres became 500-800 um in diameter and then preserved in Hibernation Medium E (Gibco BRL) for a week at 4 ℃. Those sphere were labeled by PHK26 right before the grafting. Two different type of cerebral ischemia has been prepared in host adult rats. One is middle cerebral artery occulusion by phtochemical method and the other is endothelin injecton (ET-1 ; 0.05 ng/animal) into unilateral striatum. One week following transplantation of hibernated neuroprogenitor sphere demonstrated (A) no graft survival in the core of ischemic lesion in MCA occlusion model, (B) survival of small clusters in the border of ischemic core demonstrated by faint fluorscent marker, (C) dense clusters and migrating cells in the most brain in endothelin-injected striatum. Immunostaining using anti-MAP-2 and anti-GFAP serum demonstrated neuronal as well as glial cells to the grafts. Cryopreservation in DMSO-containing medium at-180 ℃ or preservation in hibernation medium longer than one week demonstrated flagile sphere which were unable to handle for grafting surgery. This study demonstrated that neuronal progenitor cell sphere are able to survive in the brain after preservation for one week. Less
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