Molecular therapies to enhance brain recovery after experimental stroke

描述(由申请人提供)： 摘要中风是美国退伍军人的主要医疗问题之一。中风是毁灭性的，因为目前还没有可用的治疗方法来预防中风引起的神经功能障碍。预防或治疗补充omega-3多不饱和脂肪酸(n-3PUFAs)最近已成为一种非常有前途的中风神经保护策略。流行病学研究表明，n-3多不饱和脂肪酸的摄入量与缺血性中风的风险呈负相关。补充n-3多不饱和脂肪酸，尤其是DHA，可有效减轻实验性动物脑缺血损伤急性期的脑损伤程度和神经功能缺陷。然而，n-3多不饱和脂肪酸对脑损伤和中风后神经恢复的长期影响尚不清楚。我们最近创造了过度表达线虫脂肪-1基因的转基因(TG)小鼠，该基因编码一种将内源性n-6转化为n-3多不饱和脂肪酸的酶。我们对FAT-1转基因小鼠的初步研究表明，n-3多不饱和脂肪酸对缺血性中风有两个阶段(急性和延迟)的有益作用。与年龄匹配的野生型(Wt)小鼠相比，年轻(3个月大)或老年(15个月大)的FAT-1 TG小鼠对局灶性脑缺血损伤具有显著的抵抗力，表现出脑梗塞面积的缩小和神经功能的改善，持续到大脑中动脉闭塞后14天。我们发现，与Wt胎鼠相比，3个月龄的Fat-1Tg小鼠在缺血后血管生成和神经再生显著增强，这表明n-3PUFA促进中风后神经血管的再生。引人注目的是，我们发现老年小鼠中风后的血管生成和神经发生严重受损，但通过转基因表达脂肪-1几乎完全恢复。本研究试图进一步探讨n-3多不饱和脂肪酸对局灶性脑缺血的长期保护作用。目的是开发n-3多不饱和脂肪酸补充剂作为一种新的、临床可行的预防和/或治疗策略，通过刺激功能性血管和新神经元的生成来促进卒中后的长期神经康复。在目前的提案中要检验的中心假设是，预防性或治疗性n-3多不饱和脂肪酸治疗通过刺激和加强脑缺血后的修复，包括增强神经再生和血管生成，改善中风后的长期神经预后。具体目标如下：目的1.验证n-3多不饱和脂肪酸可减少幼年和老年小鼠局灶性脑缺血后长期神经功能缺陷和脑组织损伤的假说。将建立两种临床适用的方法：1)饮食递送；2)通过腺相关病毒(AAV)介导的FAT-1基因表达增加内源性n-3PUFAs的产生，以提高大脑中n-3PUFAs的水平。定量评价n-3PUFAs升高对MCAO所致缺血性脑损伤的影响。评估的终点包括功能结果、梗死面积和脑白质损伤。目的2.验证n-3多不饱和脂肪酸(n-3 PUFA)促进脑缺血后神经血管再生的假说，包括促进血管再生和神经再生。拟议的研究将定量确定n-3多不饱和脂肪酸对卒中后新生血管以及对缺血性卒中后神经干细胞的增殖、迁移、分化和神经元替换的影响。 公共卫生相关性： 叙述性中风是美国退伍军人的主要医疗问题之一。中风是毁灭性的，因为目前还没有可用的治疗方法来预防中风引起的神经功能障碍。预防或治疗补充omega-3多不饱和脂肪酸(n-3PUFAs)最近已成为一种非常有前途的中风神经保护策略。补充n-3多不饱和脂肪酸，尤其是DHA，可有效减轻实验性动物脑缺血损伤急性期的脑损伤程度和神经功能缺陷。然而，n-3多不饱和脂肪酸对脑损伤和中风后神经恢复的长期影响尚不清楚。我们的初步研究表明，n-3多不饱和脂肪酸对缺血性卒中有两个阶段(急性和延迟)的有益作用。我们发现了n-3多不饱和脂肪酸促进中风后神经血管再生的证据。因此，本提案的目标是开发n-3多不饱和脂肪酸补充剂作为一种新的、临床上可行的预防和/或治疗策略，以促进卒中后长期的神经康复。首先，我们将检验n-3多不饱和脂肪酸可以减少幼年和老年小鼠局灶性脑缺血后的长期神经功能缺陷和脑组织损伤的假设。其次，我们将检验n-3多不饱和脂肪酸治疗促进脑神经血管再生的假设。

DESCRIPTION (provided by applicant): ABSTRACT Stroke is one of the major medical concerns for United States military veterans. Stroke is devastating as currently no therapy is available to prevent stroke-induced neurological deficit. Prophylactic or therapeutic supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) has recently emerged as a highly promising neuroprotective strategy for stroke. Epidemiological studies have shown an inverse association between n-3 PUFAs intake and risk of ischemic stroke. Supplementation with n-3 PUFAs, especially DHA, effectively reduces the extent of brain damage and neurological deficits in the acute phase of ischemic injury in experimental animals. However, the long-term effect of n-3 PUFAs on brain damage and post- stroke neurological recovery is unknown. We have recently created transgenic (Tg) mice over-expressing the C elegans fat-1 gene encoding an enzyme that converts endogenous n-6 to n-3 PUFAs. Our preliminary studies with fat-1 Tg mice have suggested two-phase (acute and delayed) beneficial effects of n-3 PUFAs on ischemic stroke. The fat-1 Tg mice at either young (3-month-old) or old age (15-month-old) were remarkably resistant to focal ischemic injury compared to their age-matched wild-type (Wt) littermates, showing reduced infarct size and improved neurological functional performance up to 14 days after middle cerebral artery occlusion. We have found that angiogenesis and neurogenesis were robustly enhanced after ischemia in 3-month old fat-1 Tg mice compared to Wt littermates, suggesting that n-3 PUFAs promote post-stroke neurovascular regeneration. Strikingly, we found that post-stroke angiogenesis and neurogenesis were profoundly impaired in aged mice, but almost fully restored by transgenic expression of fat-1. This proposal attempts to further explore the long-term protective effect of n-3 PUFAs on focal cerebral ischemia. The goal is to develop n-3 PUFA supplementation as a novel, clinically feasible prophylactic and/or therapeutic strategy to promote long-term neurological recovery after stroke through stimulating the generation of functional blood vessels and new neurons. The central hypothesis to be tested in the current proposal is that prophylactic or therapeutic n-3 PUFA treatment improves long-term neurological outcomes after stroke by stimulating and enhancing post-ischemia brain repair, including augmented neurogenesis and angiogenesis. The following specific objectives are proposed: Aim 1. Test the hypothesis that n-3 PUFAs reduce long-term neurological deficits as well as brain tissue damage after focal cerebral ischemia in both young adult and aged mice. Two clinically applicable methods: 1) dietary delivery; and 2) augmented endogenous production of n-3 PUFAs via adeno-associated virus (AAV)-directed fat-1 gene expression, will be established to elevate brain levels of n-3 PUFAs. The effect of elevated n-3 PUFAs on ischemic brain injury induced by MCAO will be quantitatively evaluated. The endpoints of assessment include functional outcomes, infarct size, and white matter injury. Aim 2. Test the hypothesis that n-3 PUFA treatment enhances cerebral neurovascular regeneration, including augmented angiogenesis and neurogenesis after cerebral ischemia in both young adult and aged mice. The proposed studies will quantitatively determine the effect of n-3 PUFAs on post-stroke neovascularization and on neural stem cell proliferation, migration, differentiation and neuronal replacement following ischemic stroke. PUBLIC HEALTH RELEVANCE: NARRATIVE Stroke is one of the major medical concerns for United States military veterans. Stroke is devastating as currently no therapy is available to prevent stroke-induced neurological deficit. Prophylactic or therapeutic supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) has recently emerged as a highly promising neuroprotective strategy for stroke. Supplementation with n-3 PUFAs, especially DHA, effectively reduces the extent of brain damage and neurological deficits in the acute phase of ischemic injury in experimental animals. However, the long-term effect of n-3 PUFAs on brain damage and post-stroke neurological recovery is unknown. Our preliminary studies have suggested two-phase (acute and delayed) beneficial effects of n-3 PUFAs on ischemic stroke. We have found evidence that n-3 PUFAs promote post-stroke neurovascular regeneration. Thus, the objective of the current proposal is to develop n-3 PUFA supplementation as a novel, clinically feasible prophylactic and/or therapeutic strategy to promote long-term neurological recovery after stroke. First, we will test the hypothesis that n-3 PUFAs reduce long-term neurological deficits as well as brain tissue damage after focal cerebral ischemia in both young adult and aged mice. Second, we will test the hypothesis that n-3 PUFA treatment enhances cerebral neurovascular regeneration.