Analysis of regulation mechanism of neurogenesis after traumatic brain injury and cerebral ischemia-roles of neurotrophic factors

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1) Animal model preparation : The middle cerebral artery (MCA) occlusion model was made in mice, and neurological status was evaluated. In the animals in which MCA was permanently occluded, postoperative mortality was too high to continue further experimental analysis (mortality was about 40%). Therefore, several occlusion periods (30 to 120 min) were tried to know mortality rate of mice after surgery. Among them, 90 minutes occlusion of MCA was considered to be the best because all animals survived long enough after surgery. Evaluation of motor function using Rotor-rod showed that score declined to 50-80 % of normal value after surgery and recovered time-dependently.2) Isolation and culture of neural stem cells (NSCs) : (A) NSCs from mouse embryo brain were isolated and maintained in the culture medium. (B) neurosphere from mouse embryonic stem (ES) cells were successfully formed (see below). We precisely checked culture condition and number of spheres formed. (C) Stem cells from adip … More ose cells from mice were cultured.3) Isolation of NSCs from ES cells : We developed a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells co-cultured with the PA6 stromal cell line for at least 3 days were capable pf differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP-expressing D3-ES cells. The spheres contained nestin-positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self-renewal ability. The cells differentiated into MAP2-positive neuronal cells and GFAP-positive glial cells. gamma-Aminobutyric acid-positive cells and tyrosine hydroxylase-positive cells were also observed in the spheres. The percentages of the MAP2- or GFAP-positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue. (Kitajima H et al., 2005)4) Transplantation of above 3 kinds of NSCs into injured brain : We started to inject above 3 kinds of NSCs (A-C). Before injection, viability of some of the injected cells was pathologically confirmed. The transplanted cells are going to be pathologically examined further and motor function recovery is also to be checked. Less