Neurorecovery Effect of Novel Modified Erythropoietin on Ischemic Brain Injury

摘要 中风是美国第三大死亡原因，也是#年导致永久性残疾的最常见原因。 成年人。到目前为止，临床上唯一成功用于治疗急性中风的药物是凝块- 溶解药物组织纤溶酶原激活物(TPA)。然而，tPA必须在3小时内给药 对于不到6%的患者来说，这是一种可行的治疗方法。因此，战略 中风的治疗需要提供更广泛的患者群体。 促红细胞生成素(EPO)最近被认为是一种很有前途的神经保护和 缺血性卒中的神经康复。促红细胞生成素在体外和体内模型中都具有强大的神经保护作用 脑缺血损伤的症状。最近的一项临床试验证明了在患者中应用促红细胞生成素的有益效果 患有急性缺血性中风。因此，EPO在卒中尤其是在治疗中的神经保护作用 对于不适合tPA治疗的患者，这是一个潜在的令人兴奋的临床应用。 令人兴奋的是，我们最近的研究表明，在缺血后48小时开始给予EPO并没有 减少脑组织丢失，但刺激神经发生和少突胶质形成，减少脑白质 损伤，并显著改善缺血后的神经恢复，表明EPO是一种有前途的 中风后患者神经功能恢复的治疗试剂。然而，大剂量 卒中尤其是迟发性卒中需要多次应用促红细胞生成素 治疗。这样的EPO给药方案可能会导致多种高危因素 中风患者，如红细胞压积和血小板数量的增加，增加了发病的可能性 继发性脑梗塞。因此，减少红细胞生成活性和其他潜力的替代策略 促红细胞生成素的副作用将大大提高其在中风患者治疗中的临床应用。我们有 成功地产生了一种含有单一氨基酸突变的新型突变体EPO(MEPO)，该突变 完全缺乏红细胞生成活性。重要的是，MEPO具有神经保护作用，并保留了 刺激神经发生的能力，具有与野生型EPO类似的效果。因此，它的目标是 这项建议是为了测试缺乏红细胞生成活性的MEPO在延迟时间后的神经恢复效果 MEPO在临床相关大脑中动脉闭塞(MCAO)模型中的应用这个 提出了以下具体目标： 目的1：验证延迟给药MEPO缺乏红细胞生成活性的假说 改善缺血损伤后的神经预后。大鼠局灶性脑缺血将由 MCAO 60min。EPO和MEPO将以延迟的方式注射到小鼠体内。这些 研究将确定：1)延迟注射MEPO是否会改善神经预后 2)延迟注射MEPO是否能减少脑组织丢失。 目的2：验证MEPO通过增强神经功能而介导神经修复效应的假说 神经血管重塑和减轻脑白质损伤。我们将测试：1)是否延迟 注射MEPO促进血管生成和神经再生；2)延迟注射MEPO是否 减轻脑白质损伤和3)延迟给药MEPO是否抑制细胞死亡 缺血症。 目的3：验证MEPO介导神经发生/少突胶质形成作用的假说 EPO/？CR通过PI3K/Akt和MAPK/ERK1/2信号通路形成异源复合体。我们将测试一下 MEPO通过与EPO/？CR异源复合体结合并激活介导神经发生/少突胶质细胞发生 PI3K/Akt和MAPK/ERK1/2通路在神经球培养神经发生的体外模型中的作用 与小鼠大脑中动脉闭塞模型相同。

Abstract Stroke is the nation's third leading cause of death and the most common cause of permanent disability in adults. Thus far, the only drug that has been used successfully to treat acute stroke in the clinic is the clot- dissolving drug tissue plasminogen activator (tPA). However, tPA must be administered within 3 hours of the onset of an ischemic stroke, making it a viable treatment for less than 6% of patients. Thus, strategies providing for a wider inclusion of patient populations are needed for the treatment of stroke. Erythropoietin (EPO) has recently emerged as a promising candidate both for neuroprotection and neurorecovery in ischemic stroke. EPO has robust neuroprotective effects in both in vitro and in vivo models of ischemic injury. A recent clinical trial demonstrated the beneficial effects of administering EPO in patients with acute ischemic stroke. Thus, a neuroprotective approach using EPO in stroke, especially in treatment of patients who are not suitable for tPA treatment, represents a potentially exciting clinical application. Excitingly, our recent study indicated that EPO administration initiated at 48 hours after ischemia did not reduce brain tissue loss, but stimulated neurogenesis and oligodendrogenesis, attenuated white matter injury, and significantly improved neurological recovery after ischemia, suggesting that EPO is a promising therapeutic reagent for the neurological functional recovery of poststroke patients. However, large doses and multiple administration of EPO are required for the treatment of stroke, especially for the delayed treatment. Such a regimen of EPO administration may potentially result in multiple high risk factors for stroke patients, such as increases in hematocrit and quantity of platelets, increasing the likelihood of secondary-infarction. Accordingly, alternate strategies to reduce erythropoietic activity and other potential side effects of EPO will greatly improve its clinical applications for the treatment of stroke patients. We have successfully generated a novel mutant EPO (MEPO) containing a single amino acid mutation which completely lacks erythropoietic activity. Importantly, MEPO has neuroprotective effects and retains the ability to stimulate the neurogenesis with similar efficacies as wild-type EPO. Therefore, the objective of this proposal is to test the neurorecovery effect of MEPO lacking erythropoietic activity after a delayed administration of MEPO in the clinically relevant middle cerebral artery occlusion (MCAO) model. The following specific aims are proposed: Aim 1: To test the hypothesis that delayed administration of MEPO lacking erythropoietic activity improves neurological outcomes after ischemic injury. Focal cerebral ischemia will be induced by MCAO for 60 min. EPO and MEPO will be injected intraperitoneally into mice in a delayed manner. These studies will determine: 1) whether delayed injection of MEPO results in improved neurological outcomes after ischemia, and 2) whether delayed injection of MEPO reduces brain tissue loss. Aim 2: To test the hypothesis that MEPO mediates the neurorecovery effect via enhancing neurovescular remodeling and attenuating white matter injury. We will test: 1) whether delayed injection of MEPO enhances angiogenesis and neurogenesis; 2) whether delayed injection of MEPO attenuates white matter injury and 3) whether delayed administration of MEPO inhibits cell death after ischemia. Aim 3: To test the hypothesis that neurogenesis/oligodendrogenesis effects of MEPO is mediated by EPO/¿CR heterocomplex via PI3K/Akt and MAPK/Erk1/2 signaling pathways. We will test whether MEPO mediates neurogenesis/oligodendrogenesis via binding to EPO/¿CR heterocomplex and activating PI3K/Akt and MAPK/Erk1/2 pathways in an in vitro model of neurogenesis in neurosphere cultures as well as in the murine MCAO model.