【作者】 王芳；

【导师】 陈贵海；

【作者基本信息】 安徽医科大学 ， 神经病学， 2012， 博士

【摘要】 背景随着人类社会的老龄化，衰老相关性研究日益成为国内外研究的热点。虽然大量的资料证明衰老总是伴随着行为学的改变（尤其是认知功能减退），且许多因素（如心理社会环境、生活方式）会对衰老相关性行为产生影响，但是这些研究主要集中在学习记忆方面，且得出的结论不完全一致，同时涉及其它行为的研究很少或缺如。啮齿类动物对于衰老的研究至关重要，其中小鼠衰老模型日益受到关注。因此，探讨小鼠衰老过程中所发生的各种行为改变及其影响因素对于提出防治衰老的策略将非常有益。目的①探讨我国实验室常用的远交系CD-1小鼠中年行为学改变；②探索不同时期接受心理社会因素（应激和丰富环境）对中年CD-1小鼠行为改变的影响；③探讨桑叶活性成分1-脱氧野尻霉素（DNJ）对快速老化易感小鼠8（senescence-acceleratedpronestrains8，SAMP8）衰老相关行为改变的影响。方法根据上述目的，设计3个试验试验一：CD-1小鼠分为5月龄（青年）和12月龄（中年）组。评估小鼠衰老相关性行为改变，包括种属行为（储藏、挖掘和筑巢）、感觉运动能力（平衡木和紧绳任务）、自发性探索和焦虑行为（旷场实验、十字迷宫和黑白巷任务）、空间性学习记忆能力（六臂辐射状水迷宫和Morris水迷宫）和非空间性学习记忆能力（新物体再认）。试验二：CD-1小鼠分别于1、6和10月龄时随机分成不同处理组和空白组。不同处理组小鼠又分为应激、丰富环境和应激+丰富环境亚组。应激的小鼠每天随机接受束缚、悬吊和夜间光照方式之一，三天一循环，共四个循环。接受丰富环境的小鼠，其饲养笼内每周放置1个不同玩具，直至行为学实验结束。小鼠13月龄时评估行为学：种属行为、感觉运动能力、自发性探索和焦虑行为评估任务同试验一，空间性学习记忆能力检测用Morris水迷宫，再认记忆能力检测用新物体再认、位置再认和情景记忆任务。另设6月龄作青年组。试验三：SAMP8小鼠随机分成空白组、10mg/kg·dDNJ（低剂量）组和20mg/kg·dDNJ（高剂量）组。处理组于3月龄时开始经饮水给予不同剂量DNJ，空白组小鼠饮普通水。记录小鼠每月体重，小鼠9月龄（老年期）时行为学检测（评估内容同试验二）。另以3月龄小鼠作青年组。结果试验一：中年CD-1小鼠行为学改变中年鼠的挖掘行为和作窝能力低于青年鼠（主要是雌鼠）；平衡木成绩好于青年鼠，紧绳成绩仅中年雄鼠好。中年小鼠在旷场、十字迷宫和黑白巷试验中自发性探索活性降低和焦虑性升高（雌鼠为主）。在六臂辐射状水迷宫中，中年鼠学习期和记忆期的潜伏期和错误数均长（多）于青年鼠（雌鼠更明显）。在Morris水迷宫中，中年鼠定位航行的潜伏期显著长于青年鼠，探索原平台象限的停留时间和路程百分比显著少于青年鼠。在新物体再认任务中，中年鼠探索新物体的优先指数与青年鼠无统计学差异。试验二：应激和丰富环境对中年CD-1小鼠行为改变的影响不同时期接受应激和丰富环境对中年CD-1鼠弱化的种属行为和增强的感觉运动能力无显著影响。1月龄应激加剧中年鼠自发性探索活性的降低和焦虑性的增高，1、6月龄丰富环境可降低中年鼠的焦虑性。年龄和处理因素均未对小鼠的新物体再认和位置再认产生影响，然而，中年鼠在情景记忆任务中探索物体优先指数显著低于青年鼠，即中年鼠存在情景样记忆能力降低，不同时期应激可恶化中年鼠降低的情景样记忆，而1、6月龄应激+丰富环境和丰富环境则起到相反的作用。在Morris水迷宫中，中年鼠表现空间学习和记忆能力的下降（结果与实验一相似）。空白组小鼠的游泳路程和潜伏期显著短于1月龄应激小鼠，但长于1月龄丰富环境小鼠，1、6月龄应激小鼠的游泳路程和潜伏期显著长于同龄丰富环境小鼠，且1月龄应激小鼠也长于同龄应激+丰富环境小鼠。同时，空白组小鼠在原平台象限内停留时间和路程的百分比显著少于1、10月龄应激+丰富环境小鼠和不同时期丰富环境小鼠，不同时期应激小鼠也显著少于同龄应激+丰富环境和丰富环境小鼠。实验三：DNJ对SAMP8小鼠衰老相关行为改变的影响2个DNJ处理组体重曲线与空白组类似。老年空白组的挖掘和筑巢能力、在平衡木和紧绳上的平衡能力均显著差于青年鼠，但低剂量DNJ可改善筑巢能力，2个剂量DNJ均可减轻平衡能力下降。与青年鼠相比，空白组小鼠自发性探索活性、在旷场和高架十字迷宫中的焦虑性均显著降低，而在黑白巷中的焦虑性显著增高，低剂量DNJ可减轻这种探索活性和焦虑性的改变。空白组新物体再认（10min延迟）和位置再认（10min和24h延迟）的优先指数均显著少于青年鼠，但2个剂量的DNJ均可阻碍10min延迟新物体和位置再认成绩的下降。在情景记忆任务中，空白组优先指数显著低于青年鼠和不同剂量DNJ组。在Morris水迷宫中，空白组定位航行游泳距离和潜伏期显著长于青年鼠，在原平台象限探索时间和路程的百分比显著少于青年鼠。2个剂量的DNJ效应可纠正定位航行游泳距离的增加，低剂量DNJ可纠正探索时间和路程百分比的下降。小结1.中年CD-1小鼠存在一定的行为学改变，即种属行为、自发性探索活性和空间学习记忆能力弱化，而感觉运动能力和焦虑性增强。这些行为的改变以雌鼠为主，提示中年CD-1小鼠可能是研究衰老的良好模型。2.中年CD-1鼠的种属行为、感觉运动、新物体再认和位置再认能力可能均不受应激和丰富环境的影响，但中年期前受到应激可加重自发性探索下降、焦虑性增强、情景样记忆和空间学习记忆能力减退，而长期丰富环境可延缓这些行为改变。这些加重或延缓效应呈年龄依赖性，即越早越明显。3.长期DNJ处理不影响SAMP8小鼠生长发育，但可延缓其年龄相关性种属行为、感觉运动能力、焦虑性、空间性和非空间学习记忆能力的改变，提示DNJ具有抗脑衰老作用。结论中年期已发生了某些行为改变，但改变的方向不同，且受生活事件、社会环境的影响，时间越早效应越强。此外，生活方式或某些植物成分可延缓衰老相关性行为改变。更多还原

【Abstract】 BackgroundWith the aging of the population, there is increasing emphasis on aging-related research. Although abundant evidence indicates that aging accompanies with behavioral changes especially cognitive decline and many factors (such as psycho-social environment, lifestyles) can affect these behaviors, these studies mainly focus on learning and memory without evaluating other behaviors, moreover their conclusions are not always consistent. Rodents play a vital role in the studies of development of brain aging, and mice model has become a growing concern in age-related behavioral changes. Therefore, it is beneficial to explore the behavioral changes in the aging process and their influencing factors for finding aging interventions.Objective①To investigate the behavioral changes in the middle-aged CD-I mice that is most frequently used in Chinese laboratories;②To further investigate the effects of chronic stress and environmental enrichment on age-related behavioral changes in CD-1mice;③To explore the effects of long-term orally administered the extract of mulberry leaves i.e.,1-deoxynojirimycin (DNJ), on age-related behavioral changes in SAMP8mice.MethodsAccording to the above objective, three experiments were designed.Experiment1:Two age groups of CD-1mice (young group aged5months and middle-aged group at age of12months) were used to assess behavioral changes, including species-typical behaviors (burrowing, nesting and hoarding), sensorimotor behaviors (beam walking and tightrope task), spontaneous exploration and anxiety (open field, elevated plus maze and black-white alley), spatial learning and memory (Morris water maze (MWM) and radial six arm water maze (RAWM)) and novel object recognition.Experiment2:CD-I mice were were randomly divided into four groups as follows: normal old control,1,6and10-month-treatment. Normal old control mice did not undergo any treatment. The treatment groups were divided into stressed, stressed+enriched and enriched subgroups. The stressed mice were randomized to receive daily one way of stresses (restraint, illumination or tail suspension), three days a cycle and a totle of four cycles. The enriched mice had access to objects that stimulated their exploratory behavior and permitted social conditions until finishing the behavioral examination. The objects were rearranged every week and different objects were placed on alternate weeks for novelty. The stressed+enriched mice were exposed to stress followed by enriched environment housing. At age of13months old, all mice were conducted to behavioral evaluation, including species-typical behaviors, sensorimotor behaviors, spontaneous exploration and anxiety, spatial learning and memory (MWM) and recognition memory (novel object recognition(NOR), object recognition location (ORL) and episodic-like memory). In addition, normal6-month old mice were used as young control group.Experiment3:SAMP8mice were randomly divided into three groups:blank control,10mg/kg·d DNJ (low-dose) and20mg/kg·d DNJ (high-dose) groups. DNJ-treated mice at3months old were orally administered DNJ mixed with drinking water for6months until the behavioral tests began, and the blank control mice were received normal drinking water. At age of9months, behavioral changes were assessed as previously described in experiment2. Moreover, another3-month-old mice was set as the young control. ResultsExperiment1:The behavioral changes in middle-aged CD-1miceIn the species-typical tasks, the weights hoarded and burrowed in the middle-aged mice were significantly lower than that in the young mice, which was attributable to the female mice. The middle-aged mice showed better performance than the young mice in the beam walking, and only the middle-aged male mice had better performance than the young male mice in tightrope task. During open-field, the elevated plus maze and black-white alley testing, the middle-aged mice exhibited low locomotor activity and high anxiety, which was attributable to the female mice. In the RAWM, the latency and number of errors of the middle-aged mice were significantly longer or more than those of the young mice in both the learning and memory phases, and the cognitive impairment was mainly found in the females. In the MWM, the middle-aged mice had longer latency than the young mice but no difference in the swimming distance in the place learning test, and they showed shorter percentage of distance and time in the target quadrant than the young mice. Meawhile, the middle-aged mice showed the similar performance to the younger mice in the novel object recognition task.Experiment2:The effects of stress and enriched environment on behavioral changes in the middle-aged CD-1miceDuring the species-typical behaviors and sensorimotor testing, age effect, rather than different treatment effect, was found. In the open-field, the elevated plus maze and black-white alley tasks, the middle-aged mice showed low locomotor activity and high anxiety, and pubertal stress could aggravate the age effects but long-term enriched environment could postphone these age-related behavioral changes. In the recognition testing, there were no significant effects of age and treatment in the NOR and ORL. But, the middle-aged mice exhibited lower preferential index of exploring object than the young mice in the episodic-like memory task, indicating that the middle-aged mice had impaired episodic-like memory. And, stress experienced in different periods (in particular1and6months old) could accelerate episodic-like memory decline and enriched environment could delay this decline. In the MWM, the middle-aged mice had longer latency and swimming distance than the young mice and1-month-stressed mice, but shorter than1-month-enriched mice. The stressed mice showed longer latency and swimming distance than the enriched mice, especially in the1-and6-months stressed mice. The middle-aged mice spent less time and distance in the target quadrant than the young,1-and10-month-stresed+enriched mice, andl,6,10-month enriched mice.1,6, and10-month-stressed mice also spent more time and distance than the same age experienced stresed+enriched and enriched mice.Experiment3:The effects of DNJ on the age-related behavioral changes in SAMP8miceThere was insignificant difference of body weight between DNJ-treated group and control group. In the species-typical behaviors, the older control mice exhibited lower performance than the young mice in the nesting and burrowing, and low dose DNJ treated mice had better performance than the control mice in the nesting. In the tightrope and beam walking, the older control mice had worse performance in these tasks than the young mice, and DNJ treated mice had better performance than the control mice. During spontaneous exploration and anxiety testing, the older control mice showed low exploration activity and anxiety, and DNJ treatment could delay these behavioral changes. In the NOR task, the older control mice had lower preferential index of exploring the novel object than the young mice in the10min delay choice, and DNJ treated mice exhibited higher preferential index than the control mice. In the ORL task, the older control mice showed lower preferential index of exploring object than the young mice in the10min-and24h-delay choices, and DNJ treated mice had higher preferential index than the control mice only in the10min-delay choice. During the episodic-like memory task, the older control mice had lower preferential indexes than the young and DNJ treated mice. In the MWM, the older control mice showed longer swimming distance and latency than the young mice and shorter than DNJ treated mice in the place learning test, and they spent shorter swimming distance and time in the target quadrant than the young and low dose DNJ treated mice.Summary1. Middle-aged CD-1mouse have exhibited some behavioral changes, including low species-typical behaviors, spontaneous exploratory and spatial learning and memory, and high anxiety and sensorimotor. These behavioral changes are attributable to the females, suggesting that CD-1may be an excellent aging model.2. Stress and enrichment experienced in different period do not affect species-typical behaviors, sensorimotor, NOR and ORL in the middle-aged CD-1mice. Chronic stress experienced before middle age can aggravate some behavioral changes in middle-aged mice, including spontaneous exploratory, anxiety, episodic-like memory and spatial learning and memory. But, long-term enrichment can delay these behavioral changes. Moreover, these effects depend on onset time.3. Long-term administrated DNJ do not affect growth development in SAMP8mice, but it can delay age-related behavioral changes, including species-typical behaviors sensorimotor, anxiety, non-spatial and spatial learning and memory. Therefore, DNJ may have the property of anti-brain aging.ConclusionSome behavioral changes have occurred in middle age, but their changeable patterns are different. Moreover, life event and social environment can affect these behavioral changes. The earlier it is, the stronger the effect is. In addition, lifestyles or some food additives can delay aging-related behavioral changes.更多还原