出生前后慢性铝暴露对年轻大鼠海马LTP及nNOS、NO、Ng的影响

发布时间：2018-01-04 01:27

本文关键词：出生前后慢性铝暴露对年轻大鼠海马LTP及nNOS、NO、Ng的影响　出处：《中国医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 前言铝(aluminum)是地壳中含量最丰富的元素之一,大量蓄积可产生神经毒性作用。国内外流行病学调查和研究表明,铝易致神经元损伤,引起智力和认知能力下降等学习和记忆方面的缺陷。目前动物实验已证实,铝暴露可致大鼠痴呆,其不仅表现为学习和记忆的行为障碍,而且其病理形态学改变也与阿尔茨海默病(AD)相似。海马是学习和记忆的关键脑区,海马长时程增强(long-term potentiation,LTP)是NMDA受体依赖性突触传递效能的持续性增强,是公认的脑学习记忆功能在突触水平的研究模型和神经基础。因此研究铝暴露对海马LTP及与其突触机制有关的各项生化指标的影响,有助于从突触和蛋白分子水平阐明铝损害脑学习和记忆功能的作用机制。目前虽然铝对LTP损害作用的观察很多,但其损害作用的突触机制尚未完全阐明。母体期和断乳后发育期是脑发育和逐渐完善的重要阶段,有关此阶段铝对学习和记忆及LTP影响的报道很少,因此我们应该对铝的潜在发育毒性及直接发育毒性给予特别关注。且文献检索显示,铝对NO的直接影响尚未见报道。本实验以出生前后(包括母体期和断乳后发育期)慢性铝暴露为模型,观察铝对母体期脑发育的间接发育毒性和断乳后脑发育期的直接发育毒性所致学习和记忆能力、LTP诱导与维持的改变及对海马细胞内nNOS、NO、Ng的影响,进一步探讨铝对学习与记忆的影响及其突触机制,为铝所致儿童智力和认知能力低下性神经疾病的早期防治提供重要的实验依据。材料与方法 1、动物分组与染毒健康Wistar大鼠45只(由中国医科大学实验动物中心提供),体重300g左右,♀♂2:1。适应饲养环境1 wk后按体重随机分为3组:对照组、低剂量组和高剂量组,每组15只(♀♂2:1)。对照组(control group):饮用蒸馏水;低剂量组(0.2%-Al group):饮用含0.2%AlCl_3的蒸馏水溶液;高剂量组(0.4%-Al group):饮用含0.4%AlCl_3的蒸馏水溶液。各组自染毒始即分别合笼,子代鼠出生后通过母乳继续染毒,出生后21 d断乳,断乳后各组子代鼠对应分别以蒸馏水或母鼠染毒剂量的AlCl_3溶液喂养,至子鼠生后90 d。然后按原母鼠染毒的剂量分组,每窝取1～2只子鼠(♀♂各半),组成子代各实验组,各项实验每组7～13只,撤毒后以蒸馏水喂养。各组分批交替进行各项测试实验。动物室温度18℃～23℃,相对湿度45%～55%,光照时间12h:12h。 2、脑组织及血液中铝含量测定准确称取(0.1～0.5)g的脑组织或准确吸取(0.2-0.5)ml的全血于石英烧杯中,加(5～8)ml混酸,同时做空白对照。采用原子吸收石墨炉法测定脑铝和血铝含量。 3、学习与记忆的行为学测定测试方法为跳台法(即步下试验)。学习行为训练:将大鼠放入跳台仪内适应3 min后训练其找到平台三次,大鼠跳下平台时,立即通电给予持续电刺激(36 V)并开始计时,记录5 min内大鼠受到电刺激第一次跳上平台躲避电击的反应时间(escape latency,EL)以及受到电击的次数(number of errors)。24 h后进行记忆保持测试:记录大鼠置于平台到第1次跳下平台的时间(step-down latency,SDL)和5 min内受到电击的次数(错误次数)。 4、电生理LTP测定乌拉坦麻醉后,将动物头部固定于脑立体定位仪上,暴露颅骨。将双极刺激电极插入右侧海马CA3区锥体细胞发出的Schaffer侧枝部位(坐标:前囟后3.3mm;旁开3.8 mm;皮层下3.8 mm),将内充3 mol/L的记录玻璃微电极插入海马CA1区部位(坐标:前囟后3.3 mm;旁开1.5 mm;电极尖端抵皮层表面),然后逐步推进行细胞外记录。先找到稳定的群体锋电位(population spike,PS)后,首先记录30 min每分钟单脉冲刺激所诱发的PS。然后观察给予同样强度及波宽的的短串高频条件刺激(100 HZ、持续5s)后,记录45 min每分钟给一个单脉冲检验刺激所诱发的PS的幅值变化。 5、nNOS表达的测定电生理测试后的大鼠,4%多聚甲醛固定液心脏灌流固定。常规石蜡切片制作,用免疫组织化学的方法测定海马nNOS表达。用Metamorph/Evolution/BX51显微图像分析系统进行图像分析。 6、NO含量的测定采用硝酸还原酶法。电生理测试后的大鼠,迅速断头取脑,在冰盘上快速剖取海马,电子天平准确称重后加入9倍冷生理盐水,按试剂盒说明进行操作。 7、Ng表达的测定电生理测试后的大鼠,迅速断头取脑,在冰盘上快速剖取海马,按体积比为1:6～1:9放到预冷的裂解缓冲液中。4℃超声粉碎后12 000 r/min离心1 h,取上清分装。按照常规Western blot方法分离蛋白质。将蛋白印迹显影图扫描,再利用ChemiImager 5500 V 2103图像分析软件对实验结果(测定目标带灰度值)进行分析。 8、海马形态学超微结构观察取大鼠海马用常规透射电镜观察出生前后慢性铝暴露情况下年轻大鼠的神经细胞的形态变化。 9、统计学处理实验所得数据资料用(?)±s表示,组间资料统计用(SPSS软件)单因素方差分析(ANOVA)检验,行为学数据不服从正态分布,采用秩和检验。实验结果 1、各组大鼠血铝和脑铝含量的比较随着铝暴露剂量的升高,铝暴露组大鼠的血铝、脑铝含量逐渐升高,且均显著高于对照组(P＜0.05或P＜0.01),两铝暴露组间差异也显著(P＜0.05)。 2、各组大鼠学习和记忆行为学的改变与对照组相比,两铝暴露组大鼠第一次跳上平台的时间(反应时间)均明显延长(P＜0.01),跳下平台的潜伏期明显缩短(P＜0.01),5 min内错误次数显著增加(P＜0.01),且两铝暴露组间亦有显著性差异(P＜0.01或P＜0.05)。 3、各组大鼠PS幅值变化(即LTP增强率)的比较随着铝暴露剂量的升高,高频刺激后PS幅值增强率依次降低,两铝暴露组与对照组相比,显著降低(P＜0.01),但两暴露组之间差异不显著。 4、各组大鼠海马nNOS表达的变化与对照组相比,各铝暴露组海马CA1、CA3区nNOS表达均显著减少,且两暴露组组间也有极显著性差异(P＜0.01)。 5、各组大鼠海马细胞内NO含量的变化随着铝暴露剂量的升高,大鼠海马细胞内NO含量依次降低。两铝暴露组均明显低于对照组(P＜0.01),且两暴露组间也有极显著性差异(P＜0.01)。 6、各组大鼠海马细胞内Ng表达的变化随着染铝浓度的增加,Ng表达量降低。与对照组相比,各染毒组海马胞内Ng含量均显著降低,但两铝暴露组组间差异不显著。 7、各组大鼠海马形态学超微结构观察与对照组相比,两铝暴露组海马神经元受到损伤,细胞膜和细胞质溶解,核破损,细胞器破坏。讨论母体期和断乳后发育期脑的功能受损的研究是一个关系到婴幼儿智力健康发育的重要问题,同时,研究铝对此阶段脑发育的影响及其机制,对预防铝所致神经性疾病有着重要意义。本实验观察到,出生前后慢性铝暴露(孕期、哺乳期和断乳后发育期)的各组年轻大鼠脑铝及血铝含量均显著升高。与对照组相比,两铝暴露组的学习和记忆能力及LTP诱导与维持均明显下降,表明出生前后的铝暴露,可通过胎盘和血脑屏障进入子代大鼠体内,造成铝在子代体内蓄积,特别是脑内的蓄积,进而损害了子代鼠的学习和记忆能力。本实验还观察到,各组海马细胞内nNOS、Ng表达及NO含量也明显降低,提示,出生前后连续慢性铝暴露年轻大鼠学习记忆能力减退的可能原因之一是铝干扰了nNOS的表达,进一步抑制了NO的生成。另一原因可能是铝抑制了Ng的表达。就我们所涉猎的文献显示,铝可干扰谷氨酸能神经传递、NMDA受体相关的信号转导途径,铝可抑制参与胞内钙信号调节的多种分子途径,铝可降低小脑内钙调蛋白(CaM)的含量,铝还可使胆碱能和去甲肾上腺素能神经传递发生改变。可见,铝损伤了LTP诱导中的一系列环节,使Glu、NMDA受体、Ca~(2+)与CaM均减少,必然影响其下游过程。 Ca~(2+)/CaM可激活nNOS,铝可抑制nNOS的表达,也可通过降低Ca~(2+)浓度及CaM使nNOS激活减少,从而使NO生成减少。此外,Ng是CaM的储库,铝可抑制Ng的表达。通过上述途径导致LTP的诱导和维持受阻,致使大鼠学习和记忆能力下降。铝能够引起线粒体和DNA的损伤进而导致神经元细胞的损伤和凋亡。总之,铝可损害LTP诱导中的多种突触机制,从而损害大鼠的学习和记忆能力。结论 (1)出生前后慢性铝暴露使大鼠学习测试的反应时间显著延长,记忆测试的潜伏期明显缩短,5 min错误次数均显著增加,提示铝暴露损害了大鼠的学习和记忆行为。 (2)出生前后慢性铝暴露使PS幅值增强率减小,提示该暴露损害了大鼠LTP的诱导与维持。 (3)出生前后慢性铝暴露使海马细胞内nNOS表达减少,进而使NO含量显著降低,提示这可能是铝损害LTP的另一突触机制。 (4)出生前后慢性铝暴露使海马细胞内Ng表达减少,可能是铝损害LTP的机制之一。 (5)出生前后慢性铝暴露使脑组织的结构和细胞形态发生改变,提示铝损害了细胞功能状态可能是铝损害学习记忆的机制之一。
[Abstract]:Preface
Aluminum (aluminum) is one of the most abundant elements in the earth, a large number of stock can produce neurotoxic effects. The epidemiological investigation and study showed that Al can impair neuronal damage, cause mental and cognitive decline of learning and memory defects. The animal experiments have confirmed that aluminum exposure can cause dementia rats, which shows for behavioral disorders of learning and memory, and the pathological change and Blzheimer disease (AD).
The hippocampus is the key brain area for learning and memory, hippocampal long term potentiation (long-term potentiation LTP) is NMDA receptor dependent persistent enhancement of synaptic transmission efficiency, is recognized in the study of brain function of learning and memory at the synaptic level model and neural basis. So the research of aluminum exposure effects on the biochemical indicators of the hippocampus LTP and its synaptic mechanism, helps to elucidate the mechanism of aluminum damage brain function of learning and memory from the level of synapse and protein molecules. Although many effects of aluminum on LTP damage, but the damage of synaptic mechanism has not been fully elucidated.
Maternal and postweaning period is an important stage of brain development and gradually perfect, the effect of this stage of aluminum on learning and memory and LTP are seldom reported, so we should potential developmental toxicity and developmental toxicity of aluminum is directly given special attention. And literature retrieval shows that the direct effect of aluminum on NO has not been reported.
In this experiment, before and after birth (including the development of maternal and postweaning chronic aluminum exposure) as a model, the direct developmental toxicity caused by the ability of learning and memory were aluminum on brain development of maternal indirect developmental toxicity and weaning hindbrain development period, and maintain the change and nNOS of sea horse cells induced by LTP NO, Ng the influence to further explore the influence of aluminum on learning and memory and synaptic mechanisms, provide an important experimental basis for early prevention and treatment of mental and cognitive ability of children of low aluminum induced nerve disease.
Materials and methods
1, animal grouping and poisoning
45 healthy Wistar rats (provided by the experimental animal center of China Medical University), weighing about 300g, female male 2:1. adapt to rearing environment after 1 wk were randomly divided into 3 groups: control group, low dose group and high dose group, 15 rats in each group (female male 2:1). The control group (control group): drinking distilled water; low dose group (0.2%-Al group): distilled water drinking water containing 0.2%AlCl_3 solution; high dose group (0.4%-Al group): distilled water drinking water containing 0.4%AlCl_3 solution. Since the beginning of the exposure group were caged offspring rats after birth through breast milk exposure continued, born 21 d after weaning, weaning after the corresponding each offspring rats respectively with AlCl_3 solution or distilled water were fed the dose to 90 D. after birth, offspring and maternal exposure to the original packet according to the dose, 1~2 offspring per litter (female male half), composed of offspring in each experimental group, the experimental group with 7~13 rats in each group, after the withdrawal of drugs with distilled water feeding The test experiments were carried out alternately in each group. The temperature of the animal room was 18 to 23, the relative humidity was 45% to 55%, and the light time was 12h:12h.
2, determination of aluminum content in brain tissue and blood
The brain tissues of (0.1 to 0.5) g were accurately selected, and the whole blood of (0.2-0.5) ml was extracted accurately in the quartz beaker, plus (5~8) ml mixed acid, while blank control was performed. Atomic absorption graphite furnace method was used to determine the content of aluminum and aluminum in brain.
3, behavioural determination of learning and memory
Test method for step-down test (i.e. step test). Learning behavior training: the rats in step-down test after 3 min training to adapt to find the three platform, the rat jumped off the platform, electricity immediately given continuous electrical stimulation (36 V) and the beginning of time, rats were recorded within 5 min by electricity the reaction time of stimulating the first jump onto the platform to avoid electric shock (escape latency, EL) and the number of shocks (number of errors).24 memory test H: after rats were recorded on the platform to the first platform jump time (step-down latency, SDL) and the number of electric shocks by 5 min (the number of errors).
4, electrophysiological LTP determination
Urethane anesthesia, the head of the animal was fixed in a stereotaxic instrument, exposing the skull. Bipolar stimulating electrode was inserted into the Schaffer site of a branch of CA3 pyramidal neurons in hippocampus of the right (coordinates: anterior fontanel 3.3mm; 3.8 mm lateral cortex; 3.8 mm, 3 mol/L) filled glass microelectrode record the insertion site in CA1 region of hippocampus (coordinates: anterior fontanelle after 3.3 mm; 1.5 mm lateral; the electrode tip against the cortical surface), and then gradually push for extracellular recording. To find a stable population spike (population spike, PS), the first recorded 30 min per minute single pulse stimulation induced by PS. and observed given the same strength and the duration of the short on condition of high frequency stimulation (100 HZ, 5S), recorded 45 min per minute to the change of the amplitude of a single pulse induced by PS stimulation test.
5, determination of nNOS expression
After electrophysiological tests, 4% paraformaldehyde fixed solution was perfused into the heart. Routine paraffin sections were made. The expression of nNOS in hippocampus was detected by immunohistochemical method. Image analysis system was performed by Metamorph/Evolution/BX51 microscopic image analysis system.
6, determination of NO content
After the electrophysiological tests, the rats were quickly decapitated, and the hippocampus was rapidly dissected on the ice tray. After weighing the electronic balance accurately, 9 times cold physiological saline was added to it, and the operation was carried out according to the kit.
7, determination of Ng expression
Rats after electrophysiological testing, quickly decapitated, hippocampus quickly split in the ice tray, according to the volume ratio of 1:6 to 1:9 in lysis buffer pre cooling.4 C ultrasonic pulverization after 12000 r/min centrifugation for 1 h, the supernatant of packaging. According to the conventional Western blot method for the isolation of protein. Protein blotting the developing map scanning, using ChemiImager V 5500 analysis software on the experimental results of 2103 images (determination of target band gray value) were analyzed.
8, ultrastructural observation of hippocampal morphology
The morphologic changes of the nerve cells in the young rats were observed with conventional transmission electron microscopy (TEM) before and after the exposure to chronic aluminum exposure.
9, statistical treatment
The experimental data were expressed by (+) s. The data between groups were statistically analyzed by single factor analysis of variance (ANOVA) with SPSS software. Behavioral data did not obey normal distribution, and rank sum test was used.
experimental result
1, comparison of blood aluminum and brain Al content in rats of each group
With the increase of aluminum exposure dose, the contents of Al and Na in the aluminum exposed group increased gradually, and all were significantly higher than those in the control group (P < 0.05 or P < 0.01), and the difference between the two aluminum exposed groups was also significant (P < 0.05).
2, changes in learning and memory behavior of rats in each group
Compared with the control group, two aluminum exposure rats first jumped on the platform of the time (reaction time) were significantly prolonged (P < 0.01), jumped off the platform was significantly shortened (P < 0.01), 5 min in the number of errors increased significantly (P < 0.01), and the two groups were exposed to aluminum there is significant difference (P < 0.01 or P < 0.05).
3, comparison of PS amplitude change (LTP enhancement rate) in rats of each group
With the increase of aluminum exposure dose, the amplitude of PS amplitudes decreased after high frequency stimulation. The two aluminum exposure group was significantly lower than the control group (P < 0.01), but the difference between two exposure groups was not significant.
4, changes in the expression of nNOS in the hippocampus of rats in each group
Compared with the control group, the expression of nNOS in CA1 and CA3 areas in the hippocampus of all aluminum exposed groups decreased significantly, and there was also significant difference between the two exposed groups (P < 0.01).
5, the changes of NO content in the hippocampal cells of rats in each group
With the increase of aluminum exposure dose, the content of NO in hippocampus cells in turn decreased. The two aluminum exposed group was significantly lower than that of the control group (P < 0.01), and there was a significant difference between the two exposed groups (P < 0.01).
6, changes in the expression of Ng in the hippocampal cells of rats in each group
As the concentration of aluminum increased, the expression of Ng decreased. Compared with the control group, the Ng content in hippocampus of each exposure group was significantly reduced, but the difference between the two aluminum exposure group was not significant.
7, ultrastructural observation of hippocampal morphology in rats of each group
Compared with the control group, the hippocampal neurons in the two aluminum exposure group were damaged, the cell membrane and cytoplasm dissolve, the nucleus was damaged, and the organelles were destroyed.
discuss
Study on brain development of maternal and postweaning impairment is vital to the healthy growth of infants and young children, at the same time, influence of aluminum in stage of brain development and its mechanism, has important significance to the prevention of aluminum induced neurological diseases. This study observed that before and after birth (pregnancy, chronic aluminum exposure lactation and postweaning period) groups of young rat brain blood aluminum and aluminum content increased significantly. Compared with the control group, two of aluminum exposure on learning and memory ability and LTP group in the induction and maintenance were significantly decreased, that of aluminum exposure before and after birth, can enter offspring rats through the placenta and blood brain barrier, resulting in the aluminum accumulation in the offspring, especially the accumulation in the brain that impairs learning and memory ability of offspring rats. This experiment also observed hippocampal cells in each group nNOS, Ng expression and NO content was significantly decreased, suggesting that, One of the possible reasons for the decrease in learning and memory ability of young rats is that aluminum interferes with the expression of nNOS and further inhibits the generation of NO. Another reason is that aluminum inhibits the expression of Ng.
We read of the literature shows that aluminum can interfere with the transmission of glutamate and NMDA receptor signal transduction pathway is involved in a variety of aluminum can inhibit the molecular regulation of intracellular calcium signaling pathway, aluminum can reduce the brain calmodulin (CaM) content of aluminum can also make the cholinergic and noradrenergic neurotransmission occurs change. Obviously, LTP induced damage of aluminum in a series of links, Glu, NMDA receptor, Ca~ (2+) and CaM were reduced, will inevitably affect the downstream process.
Ca~ (2+) /CaM can activate nNOS, the expression of aluminum can inhibit nNOS, also can reduce Ca~ (2+) concentration and CaM nNOS is activated to reduce, thereby reducing NO production. In addition, Ng is the storage of CaM, the expression of Al inhibited Ng. By the way leads to the induction and maintenance of blocked LTP, resulting in the decrease of learning and memory ability in rats.
Aluminum can cause damage to mitochondria and DNA and lead to neuronal cell damage and apoptosis.
In conclusion, aluminum can damage a variety of synaptic mechanisms induced by LTP, thereby damaging the learning and memory ability of rats.
conclusion
(1) chronic aluminum exposure before and after birth significantly increased the response time of learning test, significantly shortened the latency of memory test, and significantly increased the number of erroneous errors at 5 min, suggesting that aluminum exposure impairs learning and memory behavior in rats.
(2) chronic aluminum exposure before and after birth reduced the PS amplitude enhancement rate, suggesting that the exposure damage the induction and maintenance of LTP in rats.
(3) chronic aluminum exposure before and after birth reduced the expression of nNOS in the hippocampal cells and reduced the content of NO significantly, suggesting that this may be another synaptic mechanism of aluminum damage to LTP.
(4) chronic aluminum exposure before and after birth reduces the expression of Ng in the hippocampal cells, which may be one of the mechanisms of aluminum damage to LTP.
(5) the chronic aluminum exposure before and after birth has changed the structure and cell morphology of the brain, suggesting that aluminum damages the functional state of cells, which may be one of the mechanisms that aluminum damages learning and memory.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363;R749.16

【参考文献】