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

1)动物模型制备：制作小鼠大脑中动脉（MCA）闭塞模型，评价神经功能状态。在MCA永久性闭塞的动物中，术后死亡率太高，无法继续进行进一步的实验分析（死亡率约为40%）。因此，尝试了几个闭塞期（30 - 120 min）以了解手术后小鼠的死亡率。其中，MCA闭塞90分钟被认为是最好的，因为所有动物在手术后存活足够长的时间。使用Rotor-rod进行的运动功能评估显示，手术后评分下降至正常值的50- 80%，并随时间而恢复。2）神经干细胞（NSC）的分离和培养：（A）从小鼠胚胎脑中分离NSC并在培养基中维持。(B)来自小鼠胚胎干（ES）细胞的神经球成功形成（见下文）。我们精确地检查了培养条件和形成的球体数量。(C)ADIP来源的干细胞 ...更多信息 3）从ES细胞中分离神经干细胞：我们开发了一种新的方法，通过与PA 6细胞共培养，而不是形成拟胚体，从小鼠ES细胞中诱导神经球。ES细胞与PA 6基质细胞共培养至少3天后，可分化为球形细胞。球体中的细胞均为绿色荧光蛋白（GFP）阳性，表明它们来源于表达GFP的D3-ES细胞。球体含有巢蛋白阳性细胞。与PA 6共培养时间越长，细胞球数越多。即使经过10次机械分离和继代培养，也观察到球体形成，显示其自我更新能力。细胞分化为MAP 2阳性神经元细胞和GFAP阳性胶质细胞。γ-氨基丁酸阳性细胞和酪氨酸羟化酶阳性细胞也被观察到的球体。球体中MAP 2或GFAP阳性细胞的百分比根据与PA 6细胞共培养的时间而变化。在共培养的早期阶段，产生更多的神经元，在后期，产生更多的胶质细胞。这些结果表明，神经球可以从ES细胞与PA 6共培养，这些细胞类似于神经干细胞来源于小鼠胎脑组织。（Kitajima H等人，2005）4）上述3种神经干细胞移植入损伤脑内：我们开始注射上述3种神经干细胞（A-C）。在注射前，病理学证实了一些注射细胞的活力。移植的细胞将进一步进行病理学检查，运动功能恢复也将进行检查。少

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