脑卒中是高居全球致死病因前列的重大神经系统疾病，其中缺血性脑卒中发病率最为突出。然而，目前唯一被临床公认的缺血性脑卒中治疗方法是尽早进行血液再灌注，而以往开发的神经保护剂类药物在临床实验中几乎均以失败告终。 本研究中，我们没有局限于抑制卒中早期复杂多样的细胞毒性，而是聚焦发病后数小时内出现的细胞死亡。过去的研究集中报道了细胞凋亡在缺血性脑损伤中的激活，但这种形式只存在于小部分损伤半影区内，大部分呈现坏死形态的细胞长期以来被认为是无法挽救的。近年来，一种新型程序性细胞坏死的机制及其在众多疾病中的作用逐步被揭示，但其与缺血性脑损伤的关系仍处于研究早期。为找到与临床联系更为紧密的靶点，我们首先对脑卒中患者捐献的脑组织进行研究，发现许多细胞凋亡和程序性坏死的标志性蛋白的表达及激活程度均有别于正常组织。因此，以这两种死亡形式作为靶点的新策略可能具有疾病治疗前景。 脑源性神经营养因子(BDNF)对神经组织的保护作用已被广泛报道，但其不理想的稳定性和特异性使其难以成药。因此我们开发了同样可以激活酪氨酸受体激酶B(TrkB)的抗体激动剂。通过体外实验筛选出高效激活TrkB的潜在治疗性分子Ab4B19后，我们描述了其与TrkB相互作用的特征。在鼠原代皮层神经元中，我们证明了Ab4B19可以在多种模拟缺血性损伤的细胞毒性条件下保护神经元。 在大鼠大脑中动脉梗塞／再灌注模型中，我们首先发现Ab4B19在病理条件下可在病灶区有较长时间的存留，并结合和激活主要表达在神经元上的TrkB。行为学实验证明，Ab4B19可以加速动物感觉和运动功能的恢复。这种功能的改善可能与Ab4B19有效减小脑梗塞体积相关。在分子层面研究中，我们验证了Ab4B19降低了细胞凋亡相关蛋白BAX与Bcl-2的比例并抑制了caspase-3的激活。通过检验程序性坏死标记物RIP1、RIP3及MLKL，我们发现这种死亡形式在缺血性损伤后的大脑中明显激活，且神经元是更敏感的细胞类型；而Ab4B19可能通过抑制MLKL的激活而保护神经元。最后，为促进药物向临床转化，我们在人胚胎干细胞分化的神经元中验证了Ab4B19在人源细胞中发挥的神经保护作用。 综上所述，本研究发现了细胞程序性坏死在缺血性脑损伤中的意义，并以此和细胞凋亡为靶点开发和探索了Ab4B19对该疾病治疗的潜力，为缺血性脑损伤的药物开发提供新思路。 关键词：缺血性脑损伤；受体酪氨酸激酶B；激动剂；抗体；程序性坏死

 As the second biggest killer of all human illnesses, stroke affects around 30 million people each year, with more than half being attributed to ischemic stroke. While the only FDA-approved drug treating ischemic stroke aims to achieve blood reperfusion, clinical trials employing neuroprotectants targeting single, early pathogenic mechanisms in stroke have so far been barely successful. In the present study, we focused on rescuing cell deaths at a later stage, rather than the complex cytotoxic stimuli that emerge early during stroke. In addition to the extensively studied apoptosis, recent studies reported a regulated, new type of necrosis, necroptosis, plays significant roles in stroke as well. To bridge the gap between animal research and human clinical studies, we first studied human postmortem stroke brains and found the expression and activation of both apoptotic and necroptotic proteins were altered in stroke tissues. Therefore, a new treatment plan targeting at both mechanisms might be clinically necessary. While brain-derived neurotrophic factor (BDNF) is a potent survival factor for neurons, its poor bioavailability makes it unlikely a therapeutic agent. We hence developed a TrkB agonistic antibody (Ab4B19) that mimicked BDNF functionally but exhibited better physicochemical and pharmacological features. We showed that Ab4B19 halted neuronal death in vitro under multiple conditions simulating ischemic injury. In a rat model of ischemic injury, we discovered that Ab4B19 was able to retain in the focal region and activate TrkB. Ab4B19 suppressed both apoptosis and necroptosis primarily in neurons, leading to a reduction in infarct volume and acceleration of functional recovery from sensorimotor impairments. In neurons derived from human embryonic stem cells, Ab4B19 activated TrkB and its downstream signaling, and rescued neuronal death from oxygen-glucose deprivation at a similar level as that in mouse neurons. Together, our findings may provide new insights into the mechanisms of ischemic brain injury as well as the BDNF-based therapy, and pave the way for a new strategy for ischemic stroke treatment. Key words: Ischemic brain injury; TrkB; Agonist; Antibody; Necroptosis