Combination treatment of ischemic stroke with perlecan DV and neural stem cells

项目摘要 中风是美国第五大死亡原因和长期残疾的主要原因， 治疗选择有限。即使出现了包括组织型纤溶酶原在内的再灌流疗法 激活剂(TPA)和机械血栓切除术，卒中造成的广泛损伤通常是由于缺血- 再灌流(IR)，破坏血脑屏障(BBB)，将大脑与 循环系统。IR导致BBB的两相开口，第一次发生在侮辱后几个小时内 第二次为卒中后24-74小时。后者通常是不可逆转的，因此也是最具破坏性的。 在临床上，干细胞疗法为治疗中风提供了巨大的希望，但目前的目标是中风。 在恢复期(非亚急性)通过输送细胞进行康复。在这里，我们提出了一种新的方法 在亚急性期应用神经干细胞(NSCs)以限制早期血脑屏障损伤，结果是 这将防止中风的第二阶段损害。 我们基于我们从中风小鼠中获得的广泛而新颖的初步和试验数据来提出这一建议 模型显示，IR后24小时将人(H)神经干细胞移植到脑内可改善神经功能和 减少血脑屏障的伤害。此外，我们还证明了脑细胞外基质的一个蛋白质片段 (ECM)成分Perlecan，称为结构域V(DV)，在实验性缺血性中风后具有神经保护作用，并且 可能代表着一种很有前途的新中风疗法。有趣的是，初步结果还表明，DV可以增强 神经干细胞在体外存活和分化为神经元。因此，在这项研究中，我们将检验这一假设 神经干细胞与神经保护和神经修复蛋白Perlecan DV相结合，将 协同改善中风小鼠的病理生理学和神经学结果。改进 神经干细胞移植前使用神经保护剂DV的BBB损害将改善脑环境 NSC存活，并允许更大的NSC疗效。我们将采用细丝MCAO/再灌注(IR损伤)。 模拟患者中所见的缺血性中风损伤的小鼠模型。由于衰老是中风的强烈风险因素， 我们将使用年轻成年和老年雌性和雄性小鼠，在这些小鼠中，神经行为缺陷被发现 变得更糟。目的1将确定神经干细胞和DV联合应用对亚急性卒中的影响 幼年小鼠和老年小鼠的损伤。目标2将确定亚急性神经干细胞移植的效果 以及DV联合应用于神经修复和长期中风康复。目标3将调查直接影响 Perlecan DV在a2b1诱导神经干细胞体外分化机制中的作用。 这项研究意义重大，因为它将产生新的临床前数据，证明治疗 NSC治疗，加上一种新的缺血性中风神经保护剂。这项研究将使用创新的 方法采用并结合神经干细胞的辅助药物治疗(神经保护剂)， 改善中风预后。

PROJECT ABSTRACT Stroke, the fifth leading cause of death and leading cause of long-term disability in the United States, has limited therapeutic options. Even with the advent of reperfusion therapies including tissue plasminogen activator (tPA) and mechanical thrombectomy, extensive injury from stroke often results from ischemia- reperfusion (IR), which damages the blood-brain barrier (BBB), the vessel network separating the brain from the circulatory system. IR causes biphasic openings in the BBB, the first occurring within several hours of insult and the second at 24-74 hours after stroke. The latter is generally irreversible and, thus, the most damaging. Clinically, stem cell therapy offers great promise for treating stroke, but is currently aiming for stroke rehabilitation by delivering cells during the recovery (not subacute) phase. Here, we propose a novel approach to administer neural stem cells (NSCs) in the sub-acute phase to limit early-stage BBB injuries, an outcome that would protect against the second phase of stroke damage. We base this proposal on our extensive and novel preliminary and pilot data derived from a stroke mouse model showing that human(h)NSCs transplanted into the brain 24h post-IR improves neurological function and reduces BBB damage. Further, we have demonstrated that a protein fragment of the brain extracellular matrix (ECM) component perlecan, termed domain V (DV), is neuroprotective after experimental ischemic stroke, and may represent a promising new stroke therapy. Intriguingly, preliminary results also suggest that DV enhances NSC survival and differentiation in to neurons in vitro. Therefore, in this study, we will test the hypothesis that NSCs, in combination with the neuroprotective and neuroreparative protein perlecan DV, will synergistically ameliorate pathophysiology and neurological outcome in stroked mice. Ameliorating BBB damage before NSC transplantation using a neuroprotectant DV will improve the brain environment for NSC survival and allow for greater NSC efficacy. We will employ a filament MCAO/reperfusion (IR injury) mouse model that mimics ischemic stroke injuries seen in patients. Since aging is a strong risk factor for stroke, we will use both young adult and aged female and male mice, in whom neurobehavioral deficits are found to be worse. Aim 1 will determine the effects of neural stem cells and DV co-administration on sub-acute stroke injury in young adult and aged mice. Aim 2 will determine the effects of sub-acute neural stem cell delivery and DV co-administration on neuro-repair and long-term stroke recovery. Aim 3 will investigate the direct effect of perlecan DV in mechanisms of a2b1-induced NSC neuronal differentiation in vitro. This study is significant because it will generate new preclinical data that demonstrate the optimal strategy for NSC treatment, coupled with a novel neuroprotectant for ischemic stroke. The study will use innovative methods by employing and combining adjuvant pharmacological treatment (neuroprotectant) with NSCs, to improve stroke outcome.