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孕酮对成年神经发生和脑缺血后神经再生的影响及其分子机制

Effects of Progesterone on the Neurogenesis and Neuroregeneration After Cerebral Ischemia in Adult Male Mice and Underlying Mechanisms

作者: 专业:生理学 导师:陈玲 年度:2009 学位:硕士  院校: 南京医科大学

Keywords

Progesterone (P4), dentate gyrus (DG), proliferation, survival, R4 receptor, ischemia, neurogenesis, neuroregeneration

        研究背景中枢神经系统的发育和再生一直是神经科学界倍受关注的研究领域,传统观点认为成年哺乳类动物脑组织的神经元是终极细胞,一旦遭受破坏将成为永久性的缺失。近来研究发现成年哺乳动物脑内海马齿状回(dentate dyrus,DG)和侧脑室室管膜下区(subventricular zone,SVZ)的神经干细胞和前体细胞(neural stem and progenitor cells, NSPs)能分化为神经细胞—神经发生(neurogenesis)。成年海马DG区的新生神经元与成熟的颗粒细胞相似,能与海马CA3(cornu ammonis 3 field)区神经元建立突触联系并诱发突触可塑性,提示成熟的新生神经元能发挥相应的神经功能。成年的神经发生被认为可以取代由于自然老化或病变造成的神经元死亡,最大限度地维护脑功能和结构的完整性。成年的神经发生(包括细胞增殖、迁移、分化和成熟)过程受到很多因素的调节。早期研究发现,妊娠女性脑内神经发生水平明显增高,而分娩后新生神经元的数量恢复至基础水平。由于妊娠期脑内卵巢激素包括雌二醇(estradiol,E2)和孕酮(progesterone,P4)的水平显著上升和分娩后急剧下降的特征,提示卵巢激素可能参与成年神经发生过程的调节。早期的研究已报道,E2能促进NSPs的增殖,且能促进海马DG区新生神经细胞的存活。但是,关于P4是否参与成年神经发生的调节迄今却未见报道。神经再生(neuroregeneration)是指神经系统发生损伤后脑内的神经发生过程。近来研究发现在啮齿类、灵长类动物以及人类的成年脑内某些特定区域,不仅在生理情况下存在着神经发生,而且某些神经系统的病理刺激能引起神经发生的明显增强,提出神经再生是机体对脑损伤的一种代偿性反应。很多研究已证实成年哺乳动物脑缺血能刺激DG区和SVZ的神经再生,促进新生神经元向损伤的脑区迁移。但是,实验发现脑缺血诱导的新生神经元在第2周内出现大量死亡,因此新生神经元并不能整合进入神经回路,起到修复和改善脑功能损伤的作用。脑卒中是临床上常见的急性脑血管疾病,是目前人类致残和致死的主要原因。流行病学的资料表明,绝经前女性脑卒中的发病率明显低于同龄男性,且治愈率也比男性患者高,但是绝经后女性脑卒中的发病率与同龄男性相同甚至更高,提示内源性卵巢激素的水平降低可能与脑卒中的发病率增加有关。近年来的研究已报道了E2对缺血性脑血管病的预防和治疗作用。本实验室的前期研究已证明,P4具有保护缺血所致的海马CA1区神经元死亡和改善空间记忆功能障碍的作用。但是,P4的治疗是否影响脑缺血后的神经再生目前没有明确的报道。研究目的1.研究P4对成年海马DG区神经发生过程(包括NSPs的增殖、新生神经元的迁移、分化和成熟)的调节作用及其相关的分子机制;探讨P4调节成年神经发生是否影响空间记忆功能。2.研究P4对缺血后神经再生过程(包括NSPs的增殖、新生神经元的迁移、分化和成熟)的影响及其分子机制;阐明P4调节脑缺血后的神经再生是否有助于空间记忆和运动功能损伤的恢复。实验方法第一部分:(1)用BrdU(5-溴脱氧尿嘧啶核苷)标记有丝分裂期的细胞,在BrdU注射前后连续3天(BrdU-D0-2)进行P4给药(4mg/kg/d),分别于BrdU首次给药后48小时、7天、28天和56天用免疫组化的方法检测DG区的BrdU阳性(BrdU+)细胞,以确定P4对细胞增殖(24小时-BrdU+细胞)、存活和成熟(7天、28天和56天-BrdU+细胞)的影响。(2)分别在BrdU-D0-2期、BrdU-D5-7期、BrdU-D10-12期和BrdU-D15-17期连续3天进行P4给药,观察28天-BrdU+细胞的数量,确定P4在成年神经发生过程中的调节作用。(3)通过神经元特异性核蛋白(NeuN)免疫染色,观察BrdU-D0-2期P4给药对新生神经元分化和成熟的影响。(4)阻断P4受体,阻止P4代谢物ALL的产生,抑制酪氨酸激酶(Src)、细胞外信号调节蛋白激酶(ERK1/2)和磷脂酰肌醇3-羟激酶(PI3K)的活性,探讨P4调节成年神经发生的分子机制。(5)通过Morris水迷宫的检测,探讨P4调节成年海马区的神经发生对空间记忆功能的影响。第二部分:(1)制备60分钟大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)的脑缺血再灌注动物模型。(2)在MCAO后第三天开始进行连续3次的BrdU给药,观察脑缺血对DG区和SVZ细胞增殖(24小时-BrdU+细胞)、新生神经细胞存活(28天-BrdU+细胞)、分化和成熟(28天-BrdU+/NeuN+细胞)的影响。(3)分别在BrdU-D0-2期和BrdU-D5-7期注射P4(4mg/kg/d),观察24小时和28天-BrdU+细胞,检测P4对脑缺血后神经再生的影响。(4)阻断P4受体,阻止P4的代谢,抑制Src和ERK1/2的活性,探讨P4调节神经再生过程的分子机制。(5)在BrdU给药后的56-60天通过Morris水迷宫训练,探讨P4调节神经再生对缺血脑空间记忆和运动功能损伤的恢复作用。实验结果第一部分:1.甾体激素P4能浓度依赖性地增加28天和56天-BrdU+细胞及28天-BrdU+/NeuN+细胞数量,但不改变28天-BrdU+/NeuN+细胞占BrdU+细胞总量的比例。2.与对照组相比,相同剂量的位于P4下游的其他甾体激素雄烯二醇、睾酮和雌二醇给药组的28天-BrdU+细胞数均无统计学差异;与P4给药组相比,5α-还原酶抑制剂预处理阻止P4代谢物ALL的产生后,28天-BrdU+细胞数量仍比对照组明显增多。3.与P4给药组相比,P4R阻断剂RU486、Src抑制剂PP2、ERK1/2抑制剂U0126和PI3K抑制剂LY294002预处理后,28天-BrdU+细胞数量均显著减少。4.与对照组相比,P4给药组小鼠的逃避潜伏期明显缩短,在撤台实验中,P4给药组小鼠在原平台所属象限停留时间比对照组明显延长;而P4R阻断剂RU486的预处理能阻断P4的作用。第二部分:1.与假手术组相比,脑缺血后海马DG区和SVZ的24小时和7天-BrdU+细胞明显增加;14天-BrdU+细胞数明显减少,但仍多于假手术组;56天-BrdU+细胞下降到假手术组的水平。2.与对照组相比,在MCAO后2-4天(即Brd-D0-2期)P4给药组24小时-BrdU+细胞数量明显减少;5α-还原酶的抑制剂非那司提预处理阻止P4代谢物ALL的产生后,24小时-BrdU+细胞数量明显增加。3.与对照组相比,在MCAO后7-9天(即BrdU-D5-7期)P4给药组28天-BrdU+细胞数量明显增多,并且BrdU给药后56天时的逃避潜伏期明显缩短,游泳速度明显加快。结论1. P4通过激活P4R介导的ERK-CREB信号通路和启动PI3K-Akt信号通路促进海马DG区新生神经元的存活和成熟,从而增强空间记忆功能。2. P4通过其代谢物ALL活化GABAA受体,对缺血诱导的NSPs增殖具有抑制作用;而P4通过P4R介导的ERK信号通路能促进脑缺血后新生神经元的存活,并有助于脑缺血后空间认知和运动功能损伤的恢复。本研究的结果提示:P4能促进成年的神经发生,改善更年期和老年期的记忆功能;P4能促进脑卒中后的神经再生,有助于缺血性脑损伤后认知和运动功能的恢复,具有重要的临床实用价值。
    INTRODUCTIONRecent studies have demonstrated that progenitor cells in the hippocampal dentate gyrus (DG) retain the ability to proliferate and that a significant number of the daughter cells develop into neurons in adult mammalian species—neurogenesis. Newly formed neurons, similar to established ones, are electrically active and make connections to the hippocampal CA3 field, thus suggesting that they are functional. This is reinforced by evidence that increased survival of newborn neurons is strongly correlated with hippocampus-dependent memory, while the inhibition of neurogenesis has adverse effects on hippocampus-dependent behaviors, thus implying that hippocampal neurogenesis contributes, at least in part, to learning and memory. However, little is known about the factors that regulate the processes of neurogenesis including the proliferation of neural progenitor cells and the survival of newborn cells.Past studies have revealed sex difference in the cell proliferation in the DG only when females are in a high estradiol (E2) state in comparison to that of males. The neurogenesis in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the DG significantly increases during pregnancy, and then declines immediately after parturition. Pregnancy, parturition and postpartum coincide with dramatic fluctuations in the levels of steroid hormones. Evidence indicates that in female adult rodents acute exposure to E2 initially enhances cell proliferation, and in male meadow voles E2 enhances the survival of young neurons when administered during their‘axon extension’phase. Intriguingly, P4 is naturally present at a similar level in the brains of men and women. Recent studies confirmed that the P4’s metabolites influence the process of neurogenesis. To date, however, the specific effect of P4 on adult hippocampal neurogenesis has not yet been fully elucidated.Many studies have shown that global or focal cerebral ischemia significantly increases the progenitor cell proliferation in the adult brain of gerbils, rats, mice, monkeys as well as humans. The newly proliferated cells might migrate to the damaged areas of the brain, particularly after cerebral ischemia—neuroregeneration. However, most of the newly proliferated DG cells die early, making it unlikely that these cells establish correct synaptic connections.Earlier studies have demonstrated that pre-menopausal women have a lower risk of stroke and a better outcome following stroke compared to men of the same age. Our recent study has reported a potential neuroprotective effect of P4 on ischemic brain injury, where the drug not only prevents ischemia-induced brain injury, but also reverses dysfunctions after cerebral ischemia with a longer therapeutic opportunity. Recent studies confirmed that the P4’s metabolites allopregnanolone (ALL) is a potent and highly efficacious proliferative agent in vitro and in vivo in both rodent and human neural stem cells. Our more recent study has provided evidence that P4 when administered during‘axon extension’phase enhances the survival of young neurons in male adult mice. The aim of this study was to valuate the effects of P4 on post-ischemic neurogenesis in the DG and the subventricular zone (SVZ) in the adult male mice and to explore the underlying molecular mechanisms. Earlier studies have demonstrated that pre-menopausal women have a lower risk of stroke and a better outcome following stroke compared to men of the same age. Gonadal steroid hormone P4 has also been demonstrated to exert potential neuroprotective effects in ischemic brain injury, where the drug not only prevents ischemia-induced brain injury, but also reverses dysfunctions after cerebral ischemia with a longer therapeutic opportunity. However, whether P4 modulate neuroregeneration after cerebral ischemia have not been to date investigated.OBJECTIVE1. This study investigated whether P4 influences cell proliferation and the survival of newborn neurons in the adult mice dentate gyrus, and if so, whether the P4-induced change in neurogenesis is associated with hippocampus-dependent cognitive behavior.2. The aim of this study was to explore the effects of P4 on neuroregeneration after transient middle cerebral artery occlusion (MCAO) in the subventricular zone (SVZ) and the subgranular zone (SGZ) in the DG of the adult male mice and underlying molecular mechanisms. We further investigated the relation between P4-regulated the survival of newborn neurons and P4-improved cognitive and motor deficits after stroke.METHODSPart-I: (1) Bromodeoxyuridine was used for mitotic labeling. The animals were treated with P4 at 0–2nd day (BrdU-D0-2) after the first BrdU-injection and were perfused with BrdU-staining agents at 48 hr, 7th day, 28th day and 56th day after the first injection of BrdU to investigate the effects of P4 on cell proliferation and the survival of newborn neurons. (2) To determine the administration time– dependency of P4-effect on the survival of newborn neurons, the treatment with P4 was given at BrdU-D0-2, BrdU-D5-7, BrdU-D10-12 or BrdU-D15-17, and BrdU-staining agents was examined at 28th day after the first BrdU-injection. (3) To examine the effects of P4 on the differation and maturation of newborn neurons, P4 was injected at BrdU-D0-2 and BrdU/NeuN-staining agents was examined at 28th day after the first injection of BrdU. (4) Finasteride and RU486 were given by intraperitoneal injection (i.p.). U0126, PP2, PP3 and LY294002 were infused into the cereboventricle (i.c.v.) at 30 min before P4-injection. (5) All animals were trained in the standard Morris water maze task at BrdU-D28-32 .Part-II: (1) BrdU was injected at 3rd after 60 min transient middle cerebral artery occlusion (MCAO) in the adult male mice. (2) P4 (4 mg/kg) was administered during 2–4 or 7-9 days post-MCAO. Proliferation of progenitor cell, survival and phenotypic maturation in the DG and the subventricular zone (SVZ) was assessed at 24 hr, 7th day, 14th day, 28th day and 56th day after the end of BrdU-injection. (3) Finasteride and RU486 were given by intraperitoneal injection (i.p.) and U0126 was infused into the cereboventricle (i.c.v.) at 30 min before P4-injection. (4) Morris water maze task were performed at 56-60 days post-MCAO.RESULTSPart-I: (1) The administration of P4 (4 mg/kg) at BrdU-D0-2 produced an approximately 2-fold increase in the number of 28- and 56-day-old BrdU+ cells in comparison to the controls, whereas it did not alter the number of 24/48-hr-old BrdU+ cells. (2) P4 increased the number of 28-day-old BrdU+ cells in comparison to the controls, when administered at BrdU-D5-7 but not at BrdU-D10-12 and BrdU15-17. (3) Androstenedione (Ad), testosterone (TE) or estradiol (E2) at the same-dose of P4, when administered at BrdU-D0-2, could not replicate the effect of P4, while the inhibition of 5α-reductase by finasteride did not affect the P4-action. (4) The P4R antagonist RU486 partially suppressed the P4-effect, while inhibitors for Src, MEK or PI3K totally suppressed the same effect. (5) the mice treated with P4 at BrdU-D0-2 showed a decrease in the escape-latency to the hidden-platform in comparison to the control mice; and the P4-treated mice spent more swimming time in the quadrant that previously housed the platform than the control mice did; pre-treatment with RU486 completely blocked the effects of P4 .Part-II (1) The progenitor cell proliferation were increased approximately 2-fold followed by MCAO-mice compared to sham-op, whereas the survival of newly proliferated cells were significantly decreased. (2) The administration of P4 at 2–4 post-MCAO decreased the number of 24-hour old BrdU+ cells , which was blocked by the 5α-reductase inhibitor finasteride. (3) By contrast, the administration of P4 at 7-9 days post-MCAO increased 2-fold of the number of 28-day old BrdU+ and BrdU+/NeuN+ cells in a P4 receptor (P4R) and extracellular signal-regulated kinase (ERK)-dependent manner. (4) Furthermore, compared to the MCAO-mice, the treatment with P4 improved the swim speed and decreased the escape-latency to reach the hidden-platform.CONCLUSION1. These findings in part-I suggest that P4 enhances the survival of newborn neurons through P4R and/or the Src-ERK and PI3K pathways independent of its influence on cell proliferation, which is well correlated with the potentiated spatial cognitive function of P4-treated animals.2. These results (see in part-II) indicate that the treatment with P4 after ischemia enhances functional recovery, partly through promoting survival and mature of ischemia-induced newly proliferated cells.The present study contributes to the understanding of P4 as a modulator promoting neurogenesis and neuroregeneration after cerebral ischemia in adult male mice, which may help to clarify the influence and importance of P4 in hippocampus-dependent learning and memory in the aging population, neurodegenerative disease and cerebral injury.
        

孕酮对成年神经发生和脑缺血后神经再生的影响及其分子机制

中文摘要4-8
英文摘要8-13
第一部分: 孕酮对成年海马区神经发生的作用及机制的研究14-43
    前言14-15
    材料和方法15-22
    实验结果22-25
    讨论25-28
    小结28-38
    参考文献38-43
第二部分: 孕酮对脑缺血后神经再生的作用及机制的研究43-69
    前言43-45
    材料和方法45-49
    实验结果49-51
    讨论51-55
    小结55-65
    参考文献65-69
综述69-86
    参考文献78-86
附录86-88
攻读学位期间发表文章情况88-89
致谢89
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