【作者】 张引国；

【导师】 杨卓；

【作者基本信息】 南开大学 ， 动物学， 2010， 博士

【摘要】 目的成年和老年食物限制(food restriction, FR)可以产生许多有益的生物学效应,包括延长了生命周期、延缓衰老和疾病的进程、提高学习和记忆能力及降低药物的毒性等。然而,围产期食物限制可导致胎儿宫内发育受限(intrauterine growth restriction, IUGR),主要表现为新生儿出生后体重降低、生长缓慢、学习成绩不良。实验动物研究表明,围产期食物限制或营养不良除导致体重降低、生长缓慢外,还表现为一些行为发育延迟及成年社会行为和空间学习能力下降等。由于围产期营养不良对人类的生命和健康有极大危害,因此研究其对大鼠出生后神经行为发育(发育转折点)及对认知能力的影响有利于进一步认识人类IUGR并为发展保护人类胎儿IUGR的策略提供有益的资料。本研究首先建立围产期50%食物限制(FR50) Wistar大鼠模型,之后分别观察围产期FR50对新生期子代雌雄大鼠神经行为发育(包括身体发育和神经反射)的影响。由于新生期神经行为发育的延迟或损伤往往与后期(包括幼年和成年)的认知行为下降有关,因此又研究了子代成年雄性大鼠空间学习与记忆能力、海马CA1突触可塑性及海马一氧化氮合酶(nitric oxide synthase, NOS)阳性神经元密度的变化,以揭示认知行为变化的可能机制。方法实验第一部分：围产期50%食物限制大鼠模型的建立。为了研究围产期营养不良对神经行为发育和认知能力的影响,需要有合适的动物模型。通常有蛋白限制法和食物限制法。鉴于前者在饲料制作方面困难,所以采用后者制作模型。根据食物限制的程度,通常又分为轻度(30%)、中度(50%)和重度(70%)食物限制法。本研究采用了中度食物限制法。健康成年(体重250-280 g)14周龄wistar雌鼠与雄鼠同笼(雌雄比2-3：1)。之后,每日早晨检查雌鼠阴道口或阴道涂片,发现阴道栓或阴道涂片中有大量精子存在记为大鼠妊娠第0天(embryonic day 0,E0),分笼喂养。将妊娠的雌鼠随机分为对照组(n=18)和模型组(n=16)。对照组自由摄食,并记录每日饲料消耗量。模型组从妊娠的E0到E6自由摄食,从妊娠的E7到仔鼠出生的21天(postnatal day 21, PD21)进行食物限制,食物限制量为对照组大鼠每日平均饲料消耗量的50%。两组孕鼠均予充足饮水。分别于妊娠的E1、E7、E14和E21测定孕鼠体重。大鼠自然分娩,仔鼠出生的当天记为PD0,分别于仔鼠出生的PD1、PD7、PD10、PD14和PD21称重。发现FR50新生仔鼠体重明显低于正常组新生鼠,且孕鼠E14和E21体重也明显低于正常组孕鼠。表明本实验方法模拟了围产期营养不良状态,成功复制出胎鼠IUGR,有助于下一步研究新生子代大鼠神经行为发育及围产期营养不良状态对认知能力的影响。实验第二部分：围产期食物限制对子代新生Wistar大鼠神经行为发育的影响。以肛殖距判断仔鼠的性别。典型的神经行为发育评估包括身体生长和神经反射两部分。身体生长包括开耳(PD2-PD5)、睁眼(PD14-PD17)、门齿生长(PD8-PD12)和毛发出现(PD11-PD14)。神经反射包括翻正反射(PD3-PD7)、负性趋地反射(PD6-PD10)和悬崖回避反射(PD4-PD9)。比较对照组和食物限制组新生仔鼠神经发育的变化。实验第三部分：围产期食物限制对子代成年雄性大鼠空间学习与记忆能力及海马CA1区长时程增强(long-term potentiation,LTP的影响。大鼠出生后70天(PD70).运用Morris水迷宫测试空间学习与记忆能力。Morris水迷宫测试包括定位航行实验(place navigation test)和空间探索(probe trial performance)两个部分。定位航行实验历时5天,每天分上下午两个时间段训练。。在每个时间段训练4次,分别从4个坐标象限的中点将大鼠面向池壁轻轻放入水中,记录大鼠在120 s找到平台的时间,即逃避潜伏期(escape latency)。若在120 s内找不到平台,则将大鼠引导到平台上并保持15 s。第6天撤除平台作空间探索实验,选与平台象限相对象限的中点为入水点将大鼠放入水中,记录120 s内大鼠跨越平台的次数及在目标象限的搜索时间百分比。之后,在海马CA1区在体记录场兴奋性突触后电位(fEPSP).单极记录电极垂直插入海马CA1区锥体细胞树突丛(前囟后3.4 mm,中线旁开2.5mm),双极刺激电极插入同侧海马Schaffer侧枝(前囟后4.2 mm,中线旁开3.5 mm)。调整记录电极及刺激电极深度,至记录到最大场兴奋性突触后电位(field excitatory postsynaptic potential, fEPSP)为止海马CA1区LTP区的记录参数为：以波宽0.2ms、频率0.033Hz的测试刺激,作用于大鼠海马CA3区Schaffer侧枝,诱导海马CA1区锥体细胞树突丛场兴奋性突触后电位(field excitatory postsynaptic potential, fEPSP)。以fEPSP幅度的50%电流强度作为最适刺激强度。记录30 min fEPSP后进行高频刺激。高频刺激由10串200 Hz的方波组成,串间隔2 s。之后,用相同的波宽(频率0.033Hz)记录120 min fEPSP。实验第四部分：围产期食物限制对子代成年雄性大鼠海马神经元型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)阳性细胞密度的影响。由于空间学习与记忆能力的变化及海马CA1区LTP的诱导和维持需要兴奋性神经递质谷氨酸的释放。一氧化氮(nitric oxide, NO)作为一种逆向信使,促进突触前神经元谷氨酸的释放,从而易化了在突触后神经元上记录的LTP。因此,进一步用免疫组化方法观察海马三个亚区：nNOS阳性细胞的密度。以期从一氧化氮的角度研究围产期食物限制消弱了子代空间学习和记忆能力的机制。结果1)围产期食物限制导致新生雌雄大鼠身体生长延迟,包括开耳、门齿生长、毛发和睁眼延迟。2)围产期食物限制导致雌雄新生大鼠神经反射延迟,包括翻正反射和悬崖回避反射。另外,食物限制还导致雄性新生大鼠负性趋地反射延迟,但不影响雌性新生大鼠负性趋地反射。3)围产期食物限制导致成年雄性子代大鼠空间学习与记忆能力下降。4)围产期食物限制导致成年雄性子代大鼠海马CA1区突触可塑性降低。5)围产期食物限制降低了海马三个亚区(CA1区、CA3区和齿状回)nNOS阳性细胞的密度。结论1)围产期食物限制导致新生大鼠神经发育延迟；2)围产期食物限制对新生大鼠小脑的发育可能具有性别依赖性；3)围产期食物限制可能通过抑制NO的产生降低了海马突触可塑性,从而导致空间学习与记忆能力的降低。更多还原

【Abstract】 ObjectiveFood restriction can induce the beneficial effects. It extends life span, reduces and retards the incidence of several diseases, increases learning and memory ability and attenuates the toxicological effects of drugs. Howerver, perinatal food restriction induces intrauterine growth restriction (IUGR), with neonatal babies at decreased body weight, childhood stunting and poor school achievement Perinatal food restriction or malnutrition can also induce neurodevelopmental delay, abnormal social behaviors and impaired spatial learning and memory ability besides decreased body weight and childhood stunting. Because perinatal malnutrition exerts harmful effects on human life and health, we studied the effect of perinatal food restriction on neurobehavioral development (neurodevelopmental milestones) and congnition ability. Our studies help further to recognize human fetal IUGR and provide beneficial information on developing neuroprotective therapies of human fetal IUGR. To address this question, a model of perinatal 50% food restriction (FR50) was induced in Wistar rats. The effect of perinatal FR50 on neurobehavioral development was observed, including physical growth and neurological reflexes. Since impaired or delayed neurobehavioral development is usually related to later recognition behaviors in young and adults, we studied spatial learning and memory abilities of adult offspring rats. In addition, in order to explore the possible mechanism of impaired spatial learning and memory abilities, we also observed synaptic plasticity and the density of nitric oxide synthase neurons in the hippocampus.MethodsThe first part of the experiments:Establishment of animal model with perinatal FR50. To study the effect of perinatal malnutrition on neurobehavioral development and spatial learning and memory abilities, establishment of animal model with perinatal FR50 was necessary. Generally, there are two ways, one is to restrict protein content, the other is to restrict food content. Due to difficulties of rat chow preparation on protein restriction, we apply the way of restricting food content. According to the extent of food restriction, there are three ways:slight, mild and severe food restriction. In the present study, we used the mild food restriction. Two or three healthy female rats (body weight 250-280 g,14-week old) were paired with one male (2-3:1) until mating was confirmed by observation of a copulatory plug or the presence of sperm in a vaginal rinse. The day that mating was confirmed and recorded as embryonic day 0 (E0). Each dam was singly housed. The day of birth was identified as postnatal day 0 (PDO). Afterwards, The pregnant rats were assigned radomly into two groups:the control group (n=18) and the model group (n=16). In the control group, dams had free access to diet and water during gestation and lactation (average amount of food consumed by control was recorded). In the model group, dams received normal food intake from gestation day 0 to 6, and 50% of the daily food intake of control mothers from gestation day 7 until the postnatal day 21. Water were available ad libitum in both control and FR50 rats. The body weight of dams was measured on El, E7, E14 and E21. Dams delivered spontaneously and the day of delivery was designated as PDO. The body weight of offspring rats was measured on PD1, PD7. PD10, PD14 and PD21. The result showed that FR50 reduced the body weight of newborn pups and the body weight of FR50 dams during E14 and E21. The present model mimics perinatal malnutrition and models fetal IUGR. The present study helps to further explore neurobehavioral development and cognition ability.The second part of the experiments:Effects of perinatal food restriction on neurobehavioral development in newborn Wistar rats. Gender was distinquish between male and females according to the anogenital distance. Neurodevelopmental assessment typically includes analysis of physical growth and neurological reflexes. The parameters of physical growth include timing of pinna detachment (PD2-PD5), eye opening (PD14-PD17), teeth eruption (PD8-PD12) and hair growth (PD11-PD14). Neurological reflexes include righting reflexes (PD3-PD7), negative geotaxis (PD6-PD10) and cliff avoidance reflex (PD4-PD9). The changes of neurobehavior development were observed between control and FR50 offsprings.The third part of the experiments:Effects of perinatal food restriction on spatial learning and memory ability as well as long-term potentiation in hippocampal CA1 area in adult offspring rats. On PD70, spatial learning and memory ability of rats was tested in Morris water maze (MWM). MWM task consists of two phases, place navigation test and spatial probe performance. In place navigation test, the animals were subjected to two sessions (morning and afternoon) of four trials per day for 5 consecutive days. In each trial, rats were gently released into water, facing the wall of the tank, at one of the four starting points. The trial starting points were the middle of each quadrant edge. Rats were allowed to swim freely until they climbed on the platform. The swimming time to reach the platform (escape latency from start to goal) for each trial was recorded. If they failed to locate the platform within 120s, they were placed on it for 15 seconds. After the place navigation test, the platform was removed and the same rats were used in a spatial probe test to determine memory retention. Rats were released into water at the position opposite to the target quadrant and allowed to swim for 120s. The time percent in target quadrant and the number of platform location crossings were measured. Afterwards, the in vivo field excitatory postsynaptic potential (fEPSP) was observed in the hippocampal CA1 area. Adult wistar rats were implanted with a monopolar recording electrode in the stratum radiatum of the CA1 area (3.4 mm posterior to bregma and 2.5 mm lateral to the midline), and a bipolar stimulating electrode in the Schaffer collaterals of the dorsal hippocampus (4.2 mm posterior to bregma and 3.5 mm lateral to midline) via holes drilled through the skull. The optimal depth of the electrode in the stratum radiatum of the CA1 area of the dorsal hippocampus was determined by the maximal response to the Schaffer collateral pathway stimulus. The fEPSP slope was recorded at frequence of 0.033Hz (30 s interval) by delivering a single current pulse (0.2 ms in duration) to the Schaffer collateral pathway. The stimulus intensity was adjusted to give an fEPSP amplitude of 50% of maximum. Each experiment consisted of a baseline measurement taken for 30 min, followed by a further measurement of evoked responses (120 min) after high frequency stimulation (HFS) application. HFS consisted of ten trains of 20 stimuli at 200Hz with 2 s intertrain interval, with the same stimulation intensity (0.033Hz) used for the basline recordings. fEPSP slope was analysed by Clampfit 9.0 softwale.The forth part of the experiments:Perinatal food restriction impaired spatial learning and memory behaviour and decreased the density of nitric oxide synthase neurons in the hippocampus of adult male rat offspring. Excitory neurotransmitters, such as glutamic acid, are necessary to learning and memory as well as consolidation and maintenance of LTP. Nitric oxide (NO) facilitates LTP in postsynaptic neuronal cells by promoting release of glutamic acid as a retrograde messenger. Therefore, the density of nitric oxide synthase neurons in the hippocampus of adult male rat offspring was studied using immunohistochemistry method. The aim is to study the possible mechanisms of impairment of spatial learning and memory ability by detecting the change of NO.Results1) Perinatal food restriction delayed physical growth, such as pinna detachment, hair growth, eruption of incisor teeth and eye opening. 2) Perinatal food restriction delayed neurological reflexes in surface righting reflex and cliff avoidance reflex. In addition, it also exhibited a delay in achieving negative geotaxis response in male pups but not in female pups.3) Perinatal food restriction induced spatial learning and memory deficits in adult male rat offspring.4) Perinatal food restriction impaired synaptic plasticity in hippocampal CA1 area of adult male rat offspring.5) Perinatal food restriction decreased the density of nitric oxide synthase neurons in the hippocampus (CA1 area, CA3 area and dentate gyrus) of adult male rat offspring.Conclusion1) Perinatal food restriction delayed neurobehavior development in newborn rats.2) The effect of perinatal food restriction on the development of cerebellum is gender-dependent.3) Perinatal food restriction impaired synaptic plasticity in hippocampus by reducing the content of NO. Finally, it induced spatial learning and memory deficits.更多还原