Breaking the Blood-Brain Barrier after Stroke

在美国大约有400万中风幸存者。很少有人关注脑梗死后如何替换失去的神经元和脑组织。由于其应用的时间窗口相对较宽，细胞替代疗法仍然是中风幸存者的潜在治疗选择。越来越多的证据表明，骨髓源性细胞的分化潜能不是固定的，而是受到环境因素的强烈影响。最近的研究表明，骨髓来源的干细胞分化为小胶质细胞、星形胶质细胞和神经元。脑损伤如中风可能增强这种分化潜能。与中风相关的炎症反应，虽然在某些方面是有害的，但也可能是修复性的，并为大脑提供祖细胞储存库。我们的中心假设是，在局灶性脑缺血后，骨髓来源的干细胞群体具有再生功能，并分化为脑内皮细胞、星形胶质细胞和“功能性”神经元。我们的具体目标是：确定骨髓源性干细胞是否作为脑内皮细胞的祖细胞，并有助于局灶性脑缺血损伤后发生的新生血管形成；2)。确定局灶性脑缺血后骨髓源性干细胞是否转分化为功能神经元；3)。确定静脉注射骨髓源性干细胞是否分化为缺血脑中的内皮细胞和/或功能神经元。在这个提议中，我们将使用一个辐射嵌合体，其中绿色荧光蛋白（GFP）和Y染色体标记的骨髓将被移植到受辐射的雌性小鼠中。随后，这些小鼠将接受大脑中动脉的缝合闭塞，并通过双标记免疫细胞化学确定进入大脑的标记骨髓细胞的命运。在一些实验中，表达gfp和Y染色体标记的骨髓来源细胞将被静脉注射到中风后的小鼠体内。通过脑切片，我们将确定表达神经元标记的细胞是否具有神经元的电生理和功能特征（如膜兴奋性、谷氨酸和gaba介导的突触事件）

There are approximately 4 million stroke survivors in the United States. Little attention has been directed to replacing lost neurons and brain tissue after a cerebral infarct. Since the time window for its application is relatively broad, cell replacement therapy remains a potential treatment option for stroke survivors. There is increasing evidence that the differentiation potential of bone marrow-derived cells is not fixed but strongly influenced by environmental cues. Recent studies show that bone marrow-derived stem cells differentiate into microglia, astrocytes, and neurons. Brain injury such as stroke may enhance this differentiation potential. The inflammatory response associated with stroke, while harmful in some ways, may also be reparative and serve to supply the brain with a reservoir of progenitor cells. Our central hypothesis is that after focal cerebral ischemia, a population of bone marrow-derived stem cells serve a regenerative function and differentiate into cerebral endothelial cells, astrocytes and "functioning" neurons. Our specific aims are: 1.) Determine if bone marrow-derived stem cells serve as progenitor cells for cerebral endothelial cells and contribute to the neovasculanzation that occurs after a focal cerebral ischemic insult; 2.) Determine if bone marrow-derived stem cells transdifferentiate into functioning neurons after focal cerebral ischemia; 3.) Determine if intravenously administered bone marrow derived stem cells differentiate into endothelial cells and/or functioning neurons in the ischemic brain. In this proposal we will use a radiation chimera in which Green Fluorescent Protein (GFP) and Y chromosome-tagged marrow will be transplanted into irradiated female mice. Later these mice will undergo suture occlusion of the middle cerebral artery and the fate of the tagged marrow cells that enter the brain will determined by double-label immunocytochemistry. In some experiments, GFP-expressing and Y chromosome tagged bone marrow derived cells will be administered intravenously into mice after a stroke. Using brain slices we will determine if cells which express neuronal markers have electrophysiological and functional characteristics of neurons (e.g. membrane excitability; glutamate and GABA-mediated synaptic events