摘要
以进行性认知障碍为主要临床表现的中枢神经系统退行性疾病(neurodegenerative disorders,ND)目前已成为严重影响人类健康和生活质量的主要危险因素。以阿尔茨海默病为例,目前我国患者有600万人,占全球患者总数的三分之一,每年新发病约180万人,发病形势已经十分严峻。现有的研究与防治多针对成年发病个体开展,临床上常用的如他克林、多奈哌齐等胆碱酶抑制药物都只能部分缓解认知障碍,疗效有限,提示应寻找新的策略探寻ND的发病机制与防治靶点。经过多年的探索我室成功的建立了孕期炎症刺激引发子代高血压等成年慢性疾病的动物模型(这一研究成果投送《Acta Pharmacologica Sinica》杂志,被作为特别首报,《Clinical and Experimental Pharmacology and Physiology》杂志还专门发表了述评)。在此模型基础上,我们进一步观察到母体孕期给于LPS刺激后子代大鼠3 m龄、10 m龄、20 m龄时期的学习记忆能力显著降低,并且这种降低随着年龄的增长而愈加明显,提示认知障碍的发生可能要追溯到生命早期(已发表于neuroscience)。这一发现有望将目前针对成年发病个体开展防治研究的理念提早到对生命早期,进而扩大预防和治疗的时间窗,但孕期炎症刺激影响子代认知能力的机制,诸如与认知相关的星形胶质细胞、突触、神经元在孕期炎症刺激子代动物有无改变、变化规律及其与子代动物炎症反应性的关系等重要科学问题均未阐明。为此,本课题在既往研究孕期炎症刺激引起子代认知障碍的基础上从以上几个方面较系统深入的探索性研究,旨在从新的策略探索以认知障碍为主要临床表现的中枢神经系统退行性疾病的发病新机制、寻找新的药物作用靶点以及为ND防治新途径提供新的思路和实验依据。
方法:
1.16只通过检查阴栓获得准确妊娠时间的SD孕鼠,随机分为四组:
①对照组:在孕第8-14天每天给予生理盐水0.5 ml i.p.。
②LPS组:在孕第8、10、12天给予LPS 0.79mg/kg i.p,孕第9、11、13、14天给予生理盐水。
③LPS+PDTC组:在孕第8、10、12天给予LPS 0.79 mg/kg i.p,孕第8-14天给予PDTC 100 mg/kg i.p。
④PDTC组:在孕第8-14天,每天给予PDTC 100 mg/kg i.p。
取给药后48h的孕鼠ELISA检测血清中TNF-α、IL-1β水平,观察孕期炎症刺激因素脂多糖LPS是否引起孕鼠的炎症反应以及NF-κB抑制剂PDTC作为工具药是否有效的抑制脂多糖LPS引起的炎症反应。
2.子鼠出生后监测子鼠1 m龄前的生命体征如体重、张耳、睁眼时间的改变;通过斜面转身、断崖回避、前肢悬挂、平面翻正、空中翻正、听觉惊愕、视觉定向等实验检测子鼠早期神经反射发育的情况;通过Morris水迷宫定位航行实验、平台探索实验、可视平台实验及穿梭箱实验检测子鼠早期认知能力的改变。
3.出生后25 d、3 m龄时的子鼠取血清及脑组织,ELISA检测血清及脑组织匀浆中TNF-α、IL-1β水平;血细胞计数法检测子鼠血清中白细胞总数。给予子鼠LPS (1mg/kg),检测指标同上,观测子鼠的炎症反应性。
4.子鼠出生后25 d、3 m龄时的子鼠取海马组织,4%多聚甲醛固定后石蜡切片尼氏染色观察神经组织的病理改变;戊二醛固定后透射电镜观察神经元、突触、星形胶质细胞超微结构的改变;western blot检测25 d子鼠海马组织中星形胶质细胞在发育期促进突触形成所分泌的TSP含量的变化;免疫荧光双标观察突触前膜、后膜上SYP与PSD及星形胶质细胞NF-κB与GFAP的共表达。
结果:
1.孕期腹腔注射LPS后48 h,孕鼠血清中炎症因子TNF-α、IL-1β水平仍显著高于对照组(P<0.05),而注射LPS同时给予NF-κB的抑制剂PDTC则有效降低了孕鼠血清中炎症因子TNF-α、IL-1β水平。
2.对照组、LPS组、LPS+PDTC组、PDTC组四组中每只孕鼠生育仔鼠的数量、胎鼠体重及胎鼠21 d存活率,各组之间均没有明显统计学差异(P>0.05)。
3.孕期炎症免疫刺激对子鼠早期生命体征及神经反射发育的影响:四组子鼠早期体重的增长均随着天数的增多而增加;均在9 d左右张耳、13 d左右睁眼,各种感觉反射、运动反射、协调性的发育均没有统计学差异(P>0.05)。
4.孕期炎症免疫刺激对子鼠3 m龄时学习记忆功能的影响:
1)Morris水迷宫
定位航行实验表明四组子鼠寻找平台的逃避潜伏期均随着训练时间的增多而减少,且LPS组逃避潜伏期延长,第4 d、5 d的逃避潜伏期较对照组有统计学差异(P<0.05);而空间探索实验和可视平台实验结果均无统计学差异。
2)穿梭箱
LPS组子鼠在穿梭箱试验中主动逃避反应的习得率较其余各组有所降低且被动反应的电击时间延长,与对照组子鼠相比有统计学差异(P<0.05)。
5.孕期炎症刺激对子鼠海马星形胶质细胞形态及功能的影响
1)星形胶质细胞形态学改变
LPS组子鼠海马CA1区的某些部位星形胶质细胞密集,胞体清晰,突起变长、变粗,染色深,GFAP荧光IOD值较与对照组显著增高(P<0.01)。
2)星形胶质细胞免疫功能的改变
激光共聚焦显示LPS组子鼠海马中部分星形胶质细胞的NF-κB表达较对照组增强,并从胞浆转移到核内,与蓝色结合呈紫色,荧光IOD值较对照组有显著统计学意义的升高(P<0.01)。
3)星形胶质细胞超微结构的改变
透射电镜下可见对照组、LPS+PDTC组、PDTC组星形胶质细胞核呈圆形,异染色质较多,呈小斑块状紧贴核膜内表面,偶见核仁,核周胞质内线粒体丰富。LPS组星形胶质细胞明显肿胀,细胞质溶解,出现大面积空旷区,胞内细胞器减少、消失,突起空泡化。
4)星形胶质细胞子鼠突触形成期促进突触形成功能改变
激光共聚焦检测LPS组子鼠海马中由星形胶质细胞特殊分泌的、在突触形成期必不可少的凝血酶敏感蛋白TSP的含量在25天时对照组相比表达量减少,差异有统计学意义(P<0.05);而出生后3月龄的子鼠海马中均未检测到TSP蛋白的存在。
6.孕期炎症免疫刺激对子鼠海马神经元的影响
四组子鼠出生后25天,海马CA1区神经细胞均呈多层排列,排列规则、紧密。神经细胞核呈圆形或椭圆形,核仁清晰,形态完整,未见明显的神经细胞丢失现象。到3月龄时,LPS组大鼠海马神经细胞形态完整,但数量较对照组减少,排列稀疏。透射电镜下可见四组子鼠海马CA1区神经元细胞核形态规则,核膜完整,常染色质均一,核仁明显。神经元细胞胞浆内内质网、线粒体、内质网等细胞器丰富。细胞周边可见大量的轴索。
7.孕期炎症免疫刺激对子鼠海马突触的影响
对照组、LPS+PDTC组、PDTC组子鼠海马CA1区神经毡透射电镜可见突触结构丰富,突触前膜、突触间隙、突触后膜明显,突触前终末内存在大量的突触小泡。25天时LPS组子鼠海马CA1区神经毡内可见有轻微的肿胀现象,突触前、后膜结构欠清晰,突触前终末内突触小泡明显减少;而到3月龄时,LPS组神经毡肿胀现象略有好转,但突触结构仍少于其余各组且结构不清晰,突触间隙不明显。激光共聚焦显示LPS组子鼠的突触前膜突触素蛋白SYP与突触厚膜致密物PSD表达较对照组减少。
8.孕期炎症免疫刺激对子鼠海马毛细血管的影响
对照组、LPS+PDTC组、PDTC组毛细血管内膜完整,官腔规则,偶可见内皮细胞核,基底膜无损,外周组织正常,包围毛细血管的星形胶质细胞突起完整。LPS组毛细血管内膜仍然完整,但外层与胶质细胞接触的地方肿胀,周围结构明显水肿,到3月龄时,与胶质细胞的连接有所修复,周围结构的水肿略为减轻,但与对照组相比仍存在着显著改变。
9.孕期炎症免疫刺激对子鼠炎症反应性的改变
1)基础水平
ELISA结果表明,在正常情况下孕鼠暴露于LPS对其出生后25 d、3 m龄子鼠的海马组织及血清中TNF-α、IL-1β的基础水平没有显著影响(P>0.05)。
2)炎症刺激剂刺激后反应水平
各组子鼠再次接受炎症免疫刺激2 h后,海马组织中及血清中炎症因子的水平均升高,全血中白细胞总数则呈现先降低后升高的趋势。在此过程中,LPS组的子鼠炎症因子水平较对照组显著升高且白细胞总数在8 h、24 h时仍维持在较高水平,且该炎症因子水平的升高程度与子代认知能力的降低呈显著正相关。
结论:
1.孕期炎症刺激后子代大鼠的认知能力随着年龄的增长而显著降低,但子鼠早期的生理发育和神经反射没有明显的变化,提示孕期炎症刺激后子代大鼠随着发育进程易出现认知功能的障碍。
2.孕期炎症刺激后子鼠的认知功能下降与子鼠星形胶质细胞的功能失调,神经元和突触数量减少、血脑屏障的损伤有关。
3.孕期炎症免疫刺激使子鼠全身及脑部的炎症反应处于高敏状态,这种炎症反应敏感性的增高与子代认知能力的降低呈显著正相关。
4.在孕期母体受到炎症刺激的同时用NF-κB抑制剂可有效的抑制子代神经结构的损伤,改善认知功能,为以认知障碍为主要表现的神经退行性疾病的早期防治提供了新的思路和依据。
Recently, a new concept called‘developmental basis of health and disease”have been accepted by researchers. This concept indicate that“early”or“fetal”environmental factors, particularly nutrition, program the risks for the early onset of several adult diseases such as cardiovascular and metabolic disease in adult life, including hypertension, hyperlipidemia, insulin resistance, type 2 diabetes, ischemic heart disease, breast or prostate cancer Now more and more researches focus on the relationt between maternal immune status and adult numerous neurodevelopmental disorders. The exploration is rested on epidemiological bases. Epidemiologic studies have discovered that between 2% and 5% of all live-born infants have major developmental defects and Up to 40% of the reason can be attributed to unfavorable intrauterine environment. The poor intrauterine environment can be directly or indirectly caused by maternal exposure to harmful environmental agents.
Accumulating evidence indicates that maternal infection, may leads to both a maternal and fetal inflammatory response. This response then contributes to preterm delivery, white matter damage, and other neurological disorders. With the number of preterm births is increasing, up to 10﹪of surviving infants will develop spastic motor deficits and a further 25-50﹪will suffer developmental or behavioral disabilities. However, little is known about the effect of maternal inflammation on offspring’s cognition without preterm birth. More and more people who have got neurological disorders in their adulthood were normal without conspicuous symptoms when they were born. This situation manifests our incomplete understanding of the mechanisms underlying the etiology of such neural system injuries. Therefore, further understanding of these issues is of considerable importance.
Lipopolysaccharide (LPS), a structural component of most gram- negative bacteria, is a strong inducer of innate immunity. It has been demonstrated that exposure to LPS can exacerbate neuropathology in a number of disease models such as Alzheimer’s disease, multiple sclerosis. In the last decade, animal models have demonstrated that intraperitoneal (i.p.) administration of LPS into pregnant rats has strong effects on cytokine expression in both the maternal and the fetal compartments, whether LPS crosses the placenta or not. Some cytokines, such as tumor necrosis factor (TNF), interleukin 6 (IL-6) and interleukin 1beta (IL-1-beta) have been associated with cognitive decline and dementia.
In our labtory, we had established the model that the offspring rats manifested hypertension, altered the intrarenal renin–angiotensin system, damaged renal and distinct cognitive impairment as a result of maternal exposure to the infection/ inflammation stimulus lipopolysaccharide or zymosan. We also found that NF-κB signalling may play a critical role in this process. NF-κB (p65) levels in the myocardium and kidney of offspring from the zymosan-treated group were increased significantly compared with control and greater NF-κB -binding activity was observed in the offspring of LPS-treated rats.
NF-κB is a central regulator in immunological and inflammatory processes. NF-κB exists in the cytoplasm of cells in a latent form as heterotrimers, complexed to an inhibitory subunit IκB. When the cell is activated by diverse proinflammatory stimulus, IκB is phosphorylated by the protein kinase I-kappa kinase (IκK). This allows NF-κB to translocate to the nucleus and leads to an increase in the expression of the target genes such as TNF, IL-2, IL-6, interferon-β, IL-8 and monocyte chemoattractant peptide-1 (MCP-1) and so on, ultimately induces the inflammatory response.
Obviously, all the researches mentioned above implies a hypothesis that the maternal inflammation may alter the offspring’s imflammatory response, futher impact the function of astrocyte, at last result in cognitive deficits.
Methods
1.Sixteen pregnant Sprague-Dawley (SD) rats were used. After 1 week of acclimation, pregnant rats were randomly divided into two groups: (ⅰ) a control group, administered 0.5 ml, i.p., saline on gestation Days 8, 10 and 12; (ⅱ) a LPS group, given 0.79 mg/kg, i.p. LPS at gestation Day 8, 10 and 12.
2.The serums of pregnant rats were collected at 48h after the last injection of LPS, the serum level of TNF-αand IL-1βwas detected by ELISA method.
3.Following a full term delivery, the body weight and developing landmarks of all offspring were measured until the age of 25 days and given learning and memory tests at 3 months by Morris water maze and shuttle box.
4.The pups at age 25 days and 3 months were anaesthetized with pentobarbital sodium (40 mg/kg, i.p.) and then their brains were removed and kept for following electron microscopy, double immunofluorescence and western blot assy.
5.The pups at age 25 days and 3 months were treated with LPS 1mg /kg i.p., some pups’blood samples were collected before situmulation or after situmulation 2h, 8h, 24h for cell counts by automated cell counter. Other pups were sacrificed immediately prior to treatment or 2h following stimulation. The levels of TNF-α, IL-1βin serum as well as hippocampus were measured using ELISA kits.
Results
1.The maternal serum levels of TNF-αand IL-1βwere significantly higher in LPS group than that in control group at 48 hours following LPS administration (P<0.05), while the elevated levels of cytokines was effectively inhibited by giving PDTC at the same time.
2.We had not discovered dams experienced abortion, there were no significant differences in average day of pregnancy and the number of born offspring per dam (P>0.05).
3 . During the initial post-natal development, all the pups gained body weight continuously, but did not show any differences between the four groups (P>0.05). There were no significant differences in the development landmarks ( the target time of the eruption of incisors, eys open, expanding ear) and reflective markers ( the target time of surface righting reflex, cliff avoidance, inclined plane test, overhanging, hearing shock, visual placing, space righting reflex) between four groups.
4.There were no significant difference in the spatial probe test and visible platform trial between the four groups, but the escape latency of offspring in LPS group had increased compared with control group, especially at the last two days in the training test. The offspring in LPS group in the shuttle box test aslo show small ratio of active avoidance and long electric shock time. These results indicated the impairment of spatial memory in rats of LPS group.
5.The neurons of rats in LPS group presented disordered and a clear decrease in the numbers of nuclei at 3 month. The structure of synapse became a little swollen and vacuolized along with less in number. The quantity of synaptic vesicle in synapse was decreased. We had not found obvious impairment in vascular endothelial cells, but the astrocyte arround the capillary presented serious swollen, and edema inside astrocyte cells and organelles were confirmed. We also discovered that Compared with the control, the expression of SYP and PSD decreased, the expression of GFAP and NF-κB in astrocyte was higher in the LPS group
6.in LPS group and the situation lasted in three different age stage compared with control. Differences in astrocyte morphology were also found. Cells of stellate shape with longer processes appeared in the hippocampus of offspring in LPS group.
7.There were no significant differences in the serum level of TNF-α, IL-1βand the number of WBC between four groups without immuno-inflammatory stimulus (P>0.05). After giving LPS 1mg/kg i.p., the elevation of cytokines were significantly higher and the change of WBC is more obvious in the LPS group than that in the control group. (P<0.05).
Conlusion
1.The maternal inflammation had no obvious effects on the general growth of offspring, but damage the learning and memory abilities to a certain extent at 3 month.
2.The reactions to inflammatory stimulus in offspring of LPS were more intensive than that in the control group when they suffered from immune challenge.That higher response to environment adverse factors maybe the reason of adult disease.
3.learning and memory refer to the cooperation among structures. We discovered that the maternal inflammation may effect the development of astrocyt, induced the astrocyte swollen, higher GFAP and NF-κB expression and decreased content of TSP. These changes of astrocyte further damaged the structure of synapse and capillary in the brain.
4.prenatal treated with inhibitor of the nuclear transcription factor NF-κB (pyrrolidine dithiocarbamate, PDTC) could reversed the effection of maternal inflammation on neural damage in offapring in rats.
引文
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