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， 有限的治疗选择。即使随着再灌注疗法的出现，包括组织纤溶酶原 激活剂（tPA）和机械血栓切除术，中风引起的广泛损伤通常由缺血引起- 再灌注（IR），其损害血脑屏障（BBB），血脑屏障是将脑与 循环系统。IR引起BBB中的双相开放，第一次发生在损伤的几个小时内 第二次在中风后24-74小时。后者通常是不可逆的，因此是最具破坏性的。 临床上，干细胞疗法为治疗中风提供了巨大的希望，但目前针对中风 通过在恢复（非亚急性）阶段期间递送细胞进行康复。在这里，我们提出了一种新的方法， 在亚急性期给予神经干细胞（NSC）以限制早期BBB损伤， 可以防止中风后第二阶段的损伤。 我们的基础上，我们广泛的和新颖的初步和试点数据来自中风小鼠这一建议 显示IR后24小时移植到脑中的人（h）NSC改善神经功能的模型， 减少BBB损伤。此外，我们已经证明，脑细胞外基质的蛋白质片段， (ECM)被称为结构域V（DV）的组分串珠素在实验性缺血性中风后具有神经保护作用， 可能代表了一种有前途的新中风疗法。有趣的是，初步结果还表明，DV增强了 NSC在体外存活并分化为神经元。因此，在这项研究中，我们将测试假设， 神经干细胞与神经保护和神经修复蛋白串珠素DV结合， 协同改善中风小鼠病理生理学和神经学结果。改善 在NSC移植前使用神经保护剂DV的BBB损伤将改善NSC移植的脑环境。 NSC存活率并允许更大的NSC功效。我们将采用细丝MCAO/再灌注（IR损伤） 小鼠模型，模拟在患者中观察到的缺血性中风损伤。由于衰老是中风的一个重要危险因素， 我们将使用年轻的成年和老年的雌性和雄性小鼠，在这些小鼠中发现神经行为缺陷， 变得更糟目的1将确定神经干细胞和DV联合给药对亚急性卒中的影响 年轻成年和老年小鼠的损伤。目的2将确定亚急性神经干细胞输送的效果 和DV联合给药对神经修复和长期中风恢复的影响。目标3将研究直接影响 串珠素DV在体外诱导神经干细胞分化的机制。 这项研究意义重大，因为它将产生新的临床前数据，证明最佳策略， NSC治疗，加上一种新的神经保护剂，用于缺血性卒中。该研究将采用创新的 通过使用和组合辅助药理学治疗（神经保护剂）与NSC的方法， 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.