【作者】 杨灿；

【导师】 王高华；

【作者基本信息】 武汉大学 ， 内科学， 2010， 博士

【摘要】 研究背景：很多研究强烈提示海马神经可塑性包含在应激和抑郁的病理生理学中。神经的结构和功能可以随着新的刺激改变的特性叫做神经可塑性,包括树突分支的改变,突触改变,长时程增强,轴突萌发,轴突延长,突触发生,神经发生。动态的微管是轴突和树突改变和延伸的基础。微管是基本的真核细胞支架细胞器,在形态上呈管状纤维结构,是细胞内起支撑作用的主要支架,并对细胞内物质运输起轨道和指引方向的作用。越来越多的研究表明微管结构在应激导致的神经可塑性损伤中扮演重要角色。微管主要由微管蛋白组成,微管蛋白是由α(55KD)和β(55KD)两个多肽链组成的异源二聚体,两种微管蛋白因为不同的翻译后修饰作用具有不同的亚型。目前研究比较多的α微管蛋白翻译后修饰的亚型Tyr-Tub和Acet-Tub被认为是微管动态状态的一个指标。微管系统和微管结合蛋白(microtubule-associated proteins, MAPs)相互作用,来调节微管的聚合,稳定和排列。MAPs脱磷酸化时,MAP和微管结合并促进微管的聚合和稳定。相反,当MAP磷酸化时,会诱发MAP和微管蛋白的解离,增强微管的动态性。抗抑郁药物在人们认识抑郁症的病因学以及疾病机制中起着重要的作用,最近的研究显示慢性给予抗抑郁药可以逆转应激导致的海马结构性神经可塑性的损害。氟西汀作为SSRI的典型药物,已经成为评估抑郁模型的指标性药物。研究报道称,氟西汀可以逆转应激导致的一系列改变。有关抗抑郁药物治疗对微管系统影响的研究还处在早期阶段。但是,现有的资料显示,短期和长期给药能对微管蛋白和微管动力产生影响。已有的文献显示短期给予抗抑郁药物降低微管的动态性,而长期给予抗抑郁药物比如氟西汀可以增强微管动态性。越来越多的证据显示,抑郁症最好是描述为慢性的复发性的疾病。而且,最近的研究显示,抑郁症的复燃／复发(relapse/recurrent)率十分高,大概50%重性抑郁障碍(major depressive disorder, MDD)会经历复发,并且在持续的药物治疗过程中复发和复燃的几率达到20-37%。因此,在治疗抑郁症时,预防复发和复燃成为最重要和最有挑战性的目标。就我们所知,目前关于抑郁症复发或复燃的研究多集中在临床病人,很少有文献报道研究抑郁症复发或复燃的动物模型。但由于临床研究无法更清楚了解抑郁障碍复发时脑内的神经生化,神经结构等的改变,因此,建立有关的动物模型具有积极的意义。和其他研究不同的是,本研究着力于抑郁动物模型经过药物治疗康复后,再一次接受慢性不可预见性温和应激(chronic unpredictable mild stress, CUMS),以此来模拟抑郁症的复发。目的：CUMS抑郁动物模型经氟西汀治疗后,给予再一次的CUMS,模拟抑郁症的复发,检测该模型的行为改变、脑部海马神经元的形态学改变以及细胞支架微管系统的变化。方法：(1)利用随机数字表将大鼠随机分为5组：(i)对照组：空白对照+生理盐水；(ii)CUMS组：CUMS+生理盐水；(iii)氟西汀组：CUMS+氟西汀；(iv)急性再应激组：CUMS+氟西汀+药物清洗期+急性应激；(V)CUMS再应激组：CUMS+氟西汀+药物清洗期+CUMS。(2)使用CUMS进行抑郁动物的造模,大鼠每天随机接受一种应激,持续21天。(3)大鼠接受21天CUMS后,对照组及CUMS组给予生理盐水腹腔注射,氟西汀组、急性再应激组及CUMS再应激组给予21天氟西汀治疗(10mg/kg)。(4)氟西汀治疗后,经过药物清洗期,急性再应激组暴露于一次游泳应激,CUMS再应激组大鼠再次暴露于新一轮的CUMS,以此来模拟人类抑郁症的复发。(5)在CUMS,氟西汀治疗,再次急性及慢性应激后,即第22天、第44天、第51天和第72天对大鼠进行一般情况和行为学的评估,包括体重增长、24小时摄食量、糖水偏好,旷场行为(总行程,运动速度,直立次数,粪便数)。(6)实验结束后麻醉大鼠并使用4%多聚甲醛心脏灌注,断头取脑,石蜡包埋切片,尼氏染色观察海马CA1、CA3及齿状回神经元形态学改变。(7)实验结束后麻醉大鼠断头取脑,提取总蛋白,测定总蛋白浓度,使用western blot检测α微管蛋白翻译后修饰的亚型(Tyr-Tub和Acet-Tub)表达,MAP-2以及磷酸化MAP-2表达的改变。结果：(1)CUMS后大鼠体重增长,24小时摄食量,糖水偏好,总行程,运动速度,直立次数和对照组比较均显著降低(P<0.01)。21天氟西汀治疗后,大鼠体重增长,24小时摄食量,糖水偏好,总行程,运动速度,直立次数显著增加,和对照组无统计差异,和生理盐水治疗组差异显著(P<0.01)。再次应激后,急性再应激组和氟西汀组、正常对照组比较无显著差异,CUMS再应激组和氟西汀组、对照组比较有显著差异,且CUMS再应激组和CUMS组比较体重增长,24小时摄食量,糖水偏好,总行程,运动速度各指标均有显著差异(P<0.01),两组在直立次数上虽然没有显著差异(p=0.126),但慢性再应激组的直立次数为0.125±0.3,低于CUMS组的3.75±2.05。(2)石蜡切片尼氏染色显示CUMS再应激组海马CA1区每个高倍视野下(×400)的锥体细胞计数显著低于其它各组(P<0.01)。CA3区氟西汀组,急性再应激组与正常对照组比较差异无显著性(P>0.05)。CUMS组大鼠CA3区细胞数量显著低于正常对照组(P＜0.01)。CUMS再应激组椎体细胞数不仅比正常对照组减少(P<0.01),且比CUMS组锥体细胞数量也减少(P<0.01)。齿状回区氟西汀组,急性再应激组与正常对照组比较差异无显著性(P>0.05)。而CUMS组大鼠齿状回区细胞数量显著低于正常对照组(P＜0.01)。CUMS再应激组椎体细胞数不仅比正常对照组减少(P<0.01),且比CUMS组锥体细胞数量也减少(P＜0.01)。(3) Western blot检测显示,CUMS组与对照组相比,Acet-Tub表达升高(171.84±10.3),Tyr-Tub表达降低(62.06±9.2),差异有显著性(P<0.01)；经氟西汀治疗后,Acet-Tub的表达降低(96.18±8.9),Tyr-Tub的表达升高(95.06±8.0),与对照组比较均无显著差异。急性再应激组Acet-Tub, Tyr-Tub表达与对照组均无显著差异,慢性再应激组Acet-Tub升高(244.24±8.9),Tyr-Tub表达降低(30.92±11.0),与CUMS组比较差异均有显著性。CUMS组MAP-2的表达与对照组比较无显著差异,phospho-MAP-2的表达减少(68.81±8.9),与对照组有显著差异(P<0.01)。经氟西汀治疗后,phospho-MAP-2的表达(100.60±7.3)与对照组比较无显著差异。急性再应激组Acet-Tub, Tyr-Tub, MAP-2, phospho-MAP-2表达与对照组均无显著差异,慢性再应激组phospho-MAP-2表达降低(24.75±9.7),与正常对照组及CUMS组比较差异均有显著性(P<0.01)。结论：氟西汀可逆转CUMS导致的行为,海马细胞数和细胞支架微管系统的改变。CUMS再应激组较之CUMS组有更严重的快感缺失及旷场行为的减少,同时伴随对海马细胞数及细胞支架微管系统的损害,此损害可能和MAP-2的磷酸化水平有关联。更多还原

【Abstract】 Background:Many findings strongly indicate an involvement of structural neuronal plasticity in the pathophysiology of stress and depression. The property of adult neurones to change their structure and function in response to new stimuli is called neuronal plasticity and includes changes in dendritic ramifications, synaptic remodelling, LTP, axonal sprouting, neurite extension, synaptogenesis and neurogenesis. Dynamic microtubules are fundamental for the remodelling and extension of axons and dendrites. Indeed, dynamic instability provides microtubules the capability of probing the intraneuronal space and rapidly paves the way to the eventual new synaptic partner. Microtubules are essential eukaryotic cytoskeletal organelles and growing evidence indicates that the microtubular system could play a role in stress-induced impairments in structural neuronal plasticity.Microtubules are formed by the polymerization of tubulin, a heterodimer of two subunits designated a andβ. Microtubules are functionally modified to several isoforms by post-translational modification.The expression of isoforms of a post-translational modification such as Tyr-Tub and Acet-Tub is currently used as markers of microtubule dynamics. Microtubules interact with microtubule-associated proteins (MAPs), which modulate polymerisation, stability and arrangement of microtubules. Indeed, when dephosphorylated, neuronal MAPs bind microtubules and promote tubulin assembly and microtubule stabilization. In contrast, phosphorylation of MAPs induces detachment and promotes microtubule dynamics.Antidepressant drugs have play an important role in knowing the etiology and mechanism of depression. It has been reported that chronic antidepressant treatment reverse the stress-induced decrease of hippocampal structural neuronal plasticity. Fluoxetine, as a typical antidepressant drug, is an index drug to valuate depression model. Findings have demonstrated that fluoxetine can reverse the stress-induced changes. Studies on the effects of antidepressant treatment upon the neuronal microtubular system are still in the early stages. However, current data indicate that both acute and chronic antidepressant treatments could exert an action on neuronal microtubular proteins and microtubule dynamics. Literatures indicate that a single administration of antidepressants can decrease microtubule dynamics, whereas the effect of chronic treatment such as fluoxetine enhance microtubule dynamics.There is growing evidence that depression may be best characterized as a chronic and recurrent disorder. Specifically, current estimates indicate that the rate of relapse/recurrent is very high. Approximately 50% of patients with major depressive disorder (MDD) experience recurrence, and relapse or recurrence rates are as high as 20-37% during the continuation or maintenance phase of pharmacotherapy. Prevention of recurrence and relapse is therefore one of the most important and challenging goals in the management of major depression. To the best of our knowledge, there have few literature published about the recurrence model of depression. Different from other studies, the rats in the present study were exposed to chronic unpredictable mild stress (CUMS) again after they recovered from the first CUMS induced depression, in order to simulate recurrence of depression.Objective:To investigate behavior and hippocampal morphology and cytoskeletal alterations following re-exposure to CUMS and acute swimming stress, and explore the possible mechanism.Method:(1) Animals were separated into one of five groups:control+vehicle (Control+V), chronic unpredictable mild stress+vehicle (CUMS+V), chronic unpredictable mild stress+fluoxetine (CUMS+FLX), chronic unpredictable mild stress+fluoxetine+drug washout (1 week)+acute swimming test(CUMS+FLX+AS), chronic unpredictable mild stress+fluoxetine+drug washout (1 week)+chronic unpredictable mild stress (CUMS+FLX+CUMS). (2) We used CUMS which has been shown to produce behavioral changes that are similar to human depression and considered to be a valid and useful experimental model of depression. (3) After received CUMS, rats of CUMS+V and CUMS+FLX were treated with vehicle. Rats of CUMS +FLX, CUMS+FLX+AS and CUMS+FLX+CUMS were treated with fluoxetine(10mg/kg). (4) Following fluoxetine treatment, rats of CUMS+FLX+ CUMS were re-exposed to CUMS to mimic the recurrence of depression to explore the possible alterations of cytoskeleton. Rats of CUMS+FLX+AS were re-exposure to acute swimming stress. (5) Increased weigh,24h ingestion, Sucrose preference test and open field test were assessed after CUMS, fluoxetine treatment and re-exposure to stress. (6) Nissl stain was used to observe the suvrival of the Pyramidal neurons in hippocampal CA1, CA3 and DG fields. (7) The expression of a-tubulin isoforms, MAP-2 and phospho-MAP-2 were analyzed used western blot.Results:(1) The 3 weeks of CUMS induced marked decrease in the Decreased weigh,24h ingestion, Sucrose preference test, traveled distance, moved velocity and frequencies of rearing in the stressed rats compared to the non-stressed group(p<0.01). At the end of fluoxetine treatment there were no differences between control and animals treated with fluoxetine for three weeks. In contrast, the behaviors of sucrose preference and open field were different between CUMS+V group and other groups (p<0.01 of all). There were no significantly change of sucrose preference, traveled distance, moved velocity and frequencies of rearing of CUMS+FLX+AS group following acute swimming stress. In contrast, animal treated with CUMS again consumed significantly less sucrose solution, traveled less distance and moved with less velocity in comparison with CUMS+V rats (p<0.01). The frequencies of rearing of CUMS+FLX+CUMS group were not statistically significant decreased compared to that of CUMS+V group (p=0.126). However, frequencies of rearing of CUMS+FLX+CUMS group were deduced to 0.125±0.354, lower than 3.75±2.053 of CUMS+V group. (2) The Pyrmaidal neurons in hippocampal CA1 region in CUMS+FLX+CUMS group were were singificantly decreased compare to other groups (p<0.01). The amount of neurons in hippocampal CA3 and DG region have no difference between CUMS+V, CUMS+FLX and CUMS+FLX+AS. The amount of neurons in hippocampal CA3 and DG region in CUMS+V was significantly less than that of Control+V group(p<0.01) whereas the amount of neurons in CUMS+FLX+CUMS was significantly less than that of CUMS+V group(p<0.01). (3) The densitometric analyses of the Acet-Tub expression of CUMS+V group showed a significant increase (P＜0.01) to 172±11% in rats submitted to CUMS and the Acet-Tub expression of CUMS+FLX+CUMS group increased significantly (P＜0.01) to 239±10% following re-exposure to CUMS. Furthermore, the post hoc analysis also showed there was significant deference between CUMS+V and CUMS+FLX+CUMS group (P<0.01, Bonferroni test). The densitometric analyses also showed that Tyr-Tub expression of CUMS+V group was significantly decreased to 61±11% following CUMS stress and the post hoc analysis revealed a significant (P<0.01) difference compared with other groups. Tyr-Tub expression of CUMS+FLX+CUMS group significantly decreased to 31 ±7% following re-exposure to CUMS, difference significantly (P＜0.01) compared with other groups, including the CUMS+V group. In contrast, the a-tubulin isoforms expression of animals treated with fluoxetine and animals received acute swimming stress did not differ statistically from the Control+V group (p>0.05 for both). The one-way ANOVA performed on levels of the hippocampal MAP-2 indicated that the staining of MAP-2 by MAP-2 antibody did not significantly alter after stress or re-exposure to stress while levels of the hippocampal phospho-MAP-2 altered significantly after stress and re-exposure to stress. The densitometric analyses of the phospho-MAP-2 expression showed a significant decrease (P＜0.01) to 64±9% in CUMS+V rats submitted to CUMS and a significant decrease (P<0.01) to 22±11% following re-exposure to CUMS in CUMS+FLX+CUMS group. In particular, the phospho-MAP-2 expression of CUMS+FLX rats and CUMS+FLX+AS rats did not differ statistically from the Control+V group (p>0.05 for both).Conclusion:Our results suggest that CUMS and fluoxetine affect microtubule dynamics in the hippocampus. These effects appear to be mediated by the degree of phosphorylation of MAP-2. Furthermore, the stressed rats were more sensitive to the subsequent CUMS and their hippocampal cytoskeleton became more impaired. It suggests a possible role of cytoskeletal proteins in mediating functional and/or morphological changes in rat hippocampus and in the recurrence/relapse of depression.更多还原