Neurovascular protection of the brain following ischaemic stroke and the role of the hamartin/mTOR pathway

大脑由血液提供氧气和葡萄糖，大脑内的细胞将氧气和葡萄糖转化为能量来维持大脑功能。因此，持续的血液供应至关重要，因为当流向大脑的血液流动受阻时，如中风，可能会发生严重的脑损伤。中风是第三大致死原因和第一大致残原因，每年给英国经济造成37亿英镑的损失。中风只有一种批准的治疗方法可以分解血栓，只有不到15%的患者有资格接受这种治疗。因此，发现治疗中风的新策略是必要的。重要的是，中风的一个主要后果是血脑屏障(BBB)的破坏，这是一种阻止物质从血液进入大脑的物理屏障。这种血脑屏障的分解会导致肿胀并损害脑细胞。许多类型的细胞形成并维持血脑屏障。这组细胞统称为神经血管单位(NVU)，在控制大脑内的血液流动方面也很重要。人们正在尝试寻找治疗中风的药物靶点，以保护神经元(大脑的主要细胞类型)并保存NVU功能(保持血脑屏障关闭和维持血液流动)。我们认为其中一个靶点是细胞中的一条生化信号通路，称为Hamartin/哺乳动物雷帕霉素靶点(MTOR)通路。Hamartin是一种蛋白质，它可以阻止另一种名为mTOR的蛋白质的活性，从而阻止细胞生长和其他细胞功能。我们最近的研究表明，增加Hamartin可以保护中风后的神经元。我们也有初步数据表明，增加Hamartin可以保护内皮细胞(一种重要的血流和BBB功能细胞类型)。MTOR通路与衰老、痴呆和血流减少有关，因此阻断mTOR可能是中风后保护大脑的有用策略。这项建议希望回答5个问题：1)大鼠中风后脑内hamartin/mTOR途径的活性是否发生变化？2)增加hamartin或阻断mTOR能否保护中风后的大脑？3)在正常情况下和中风后阻断mTOR是否会改变脑中的血液流动？4)当阻断mTOR时，哪些单个细胞类型负责保护大脑？5)人类中风后脑内hamartin/mTOR途径的活性是否发生变化？为了回答问题1，将评估发生实验性中风的大鼠大脑中hamartin/mTOR途径的蛋白质变化。为了回答第二个问题，发生实验性中风的大鼠将接受一种病毒，这种病毒会增加大脑中的Hamartin水平，而其他大鼠将接受阻断mTOR的药物。我们希望增加Hamartin或阻断mTOR将减少中风后对大脑的损害。为了回答问题3，我们将给普通大鼠服用mTOR阻滞剂，看看在正常情况下和大脑受到刺激时，这是否可以改变血流。然后，我们将观察mTOR阻滞剂是否也会改变中风后的血流量。然后问题4将回答mTOR在哪些细胞中起保护作用。不同的细胞将从老鼠的大脑中单独分离出来，并暴露在缺乏氧气和葡萄糖的环境中，模拟中风。病毒会增加Hamartin的含量，或者药物会阻止mTOR，这会告诉我们哪些脑细胞受到保护。为了回答问题5，将对已故人类中风患者的大脑进行hamartin/mTOR途径蛋白质的变化评估。这将表明，这一途径在人类和大鼠中都受到中风的影响。我们希望这些研究将揭示阻断mTOR通路是否会对大脑提供保护，进一步发展后，可能会为中风患者提供一种新的治疗选择。

The brain is supplied with oxygen and glucose delivered by the blood, and cells within the brain convert that oxygen and glucose into energy to maintain brain function. Therefore, it is critical that blood supply is continuously available, because when blood flow to the brain is blocked such as in stroke, severe brain damage can occur. Stroke is the third leading cause of death and leading cause of disability costing the UK economy £3.7 billion per year. Stroke has only one approved treatment which breaks down blood clots and less than 15% of patients are eligible for this treatment. Therefore, discovering novel strategies for stroke treatment is warranted. Importantly, a major consequence of stroke is the breakdown of the blood-brain barrier (BBB), a physical barrier that prevents passage of material from the blood to the brain. Breakdown of this BBB causes swelling and damages brain cells. Many types of cells form and maintain the BBB. Collectively this grouping of cells is called the neurovascular unit (NVU), and is also important in controlling blood flow within the brain. Attempts are being made to find drug targets for stroke treatment that protect neurons (the brain's major cell type) and conserve NVU function (keeping the BBB closed and maintaining blood flow). We believe one such target to be a biochemical signalling pathway in cells called the hamartin/mammalian target of rapamycin (mTOR) pathway. Hamartin is a protein that acts to block the activity of another protein called mTOR, resulting in the prevention of cell growth and other cell functions. We recently showed that increasing hamartin can protect neurons after stroke. We also have preliminary data that shows that increasing hamartin can protect endothelial cells (an important cell type for blood flow and BBB function). The mTOR pathway has been linked with ageing, dementia and decreased blood flow, so blockade of mTOR may be a useful strategy for protection of the brain following stroke. This proposal hopes to answer 5 questions:1) Does the activity of the hamartin/mTOR pathway change in the brain after stroke in rats?2) Can increasing hamartin or blocking mTOR protect the brain after stroke?3) Does blocking mTOR change blood flow in the brain under normal conditions and following stroke?4) Which individual cell types are responsible for protection of the brain when blocking mTOR after stroke?5) Does the activity of the hamartin/mTOR pathway change in the brain after human stroke?To answer question 1, brains from rats that have had an experimental stroke will be assessed for changes in proteins of the hamartin/mTOR pathway. To answer question 2, rats that had an experimental stroke will receive a virus that increases hamartin levels in the brain, while other rats will receive drugs that block mTOR. We hope that increasing hamartin or blocking mTOR will reduce damage to the brain after stroke. To answer question 3, we will give ordinary rats mTOR blockers to see if this can alter blood flow under normal conditions and when the brain is stimulated. We will then see if mTOR blockers also change blood flow after stroke. Question 4 will then answer in what cells is mTOR being protective. Different cells will be individually isolated from rat brains and exposed to an environment that lacks oxygen and glucose, mimicking a stroke. Hamartin will be increased with a virus or mTOR will be blocked with drugs, and this will tell us which brain cells are protected. To answer question 5, brains from deceased human stroke patients will be assessed for changes in proteins of the hamartin/mTOR pathway. This will show that this pathway is affected by stroke in humans as well as rats. We hope that these studies will reveal whether blocking the mTOR pathway will provide protection to the brain, which after further development, could provide a novel treatment option for stroke patients.