APP/PS1转基因小鼠学习记忆能力下降与胼胝体脱髓鞘损伤的关系

目的探讨12月龄APP/PS1转基因小鼠学习记忆能力下降与胼胝体脱髓鞘损伤之间的关系。方法 12只12月龄APP/PS1转基因小鼠为AD组,12只同窝阴性小鼠为野生组。Morris水迷宫检测两组学习记忆能力;卢卡斯快蓝染色观察胼胝体神经纤维形态;免疫组化检测胼胝体髓鞘碱性蛋白(MBP)含量;硫黄素S染色观察淀粉样斑块在胼胝体的沉积情况。结果与野生组相比,AD组逃避潜伏期明显延长(Z2.873,P0.01),穿越平台次数显著减少(t=-7.339,P0.001)。AD组胼胝体神经纤维髓鞘稀疏,排列紊乱,出现较多空洞;MBP含量较野生组显著降低(t=-4.481,P0.001);胼胝体区出现淀粉样斑块沉积。结论 12月龄APP/PS1转基因小鼠学习记忆能力下降,可能与淀粉样斑块在胼胝体的沉积引起胼胝体神经纤维脱髓鞘损伤有关。     

小续命汤对APP/PS1转基因鼠海马区Aβ、GFAP蛋白的影响及其行为学分析

目的:观察小续命汤对APP/PS1转基因鼠海马区Aβ及GFAP蛋白的影响,探讨小续命汤改善认知功能障碍的作用机制。方法:采用APP/PS1双转基因小鼠建立阿尔茨海默病模型,将同月龄的同窝阴性鼠作为空白对照组,按照随机数字表将基因鼠分为模型组和小续命汤组,模型组和空白对照组不给药,小续命汤组按照13 g/kg连续灌胃90 d。Morris水迷宫法检测小鼠学习记忆能力,免疫组化染色观察3组小鼠大脑海马区Aβ、GFAP蛋白的表达。结果:与模型组比较,小续命汤组在水迷宫空间探索实验中,到达目标区域的时间及搜索距离明显缩短(P0.05),穿梭次数明显增多(P0.05);免疫组化结果显示:与空白对照组比较,模型组小鼠大脑海马区Aβ阳性蛋白及GFAP阳性细胞均明显增加(P0.01);与模型组比较,小续命汤组Aβ蛋白的沉积及GFAP阳性细胞均明显减少(P0.05)。结论:小续命汤改善APP/PS1基因鼠的认知功能可能与减少大脑海马区Aβ沉积、降低星形胶质细胞的活化有关。     

人羊膜间充质干细胞移植对阿尔茨海默病转基因小鼠的行为学和β-淀粉样蛋白的影响

背景:研究发现APP基因与阿尔茨海默病发病密切相关。β-淀粉样蛋白是阿尔茨海默病患者脑内老年斑的主要成分,基因突变和外界环境的影响能破坏β-淀粉样蛋白的动态平衡,从而引发或加速阿尔茨海默病的产生和发展。目的:探讨人羊膜间充质干细胞尾静脉移植对阿尔茨海默病转基因小鼠学习记忆能力及脑组织β-淀粉样蛋白表达的影响。设计、时间及地点:随机对照动物实验,于2008-05/10在郑州大学微生物学与免疫学教研室和河南省中医药治疗研究院完成。材料:健康剖宫产产妇志愿捐献的羊膜,由郑州大学第一附属医院产科提供。APP转基因鼠29只,PCR技术鉴定转APP-基因小鼠9只,作为正常组;转APP 基因小鼠20只,随机分为细胞移植组、对照组,10只/组。方法:无菌条件下体外分离培养人羊膜间充质干细胞,传至第3代将细胞浓度调整为1×109L-1,经尾静脉注入0.5mL至细胞移植组小鼠体内;对照组经尾静脉注入同体积的生理盐水;正常组小鼠不给予任何干预措施。主要观察指标:采用Morris水迷宫测定小鼠逃避潜伏期、穿越平台次数及在平台象限的时间,刚果红染色观察小鼠脑组织内β-淀粉蛋白的表达。结果:定位航行试验中,移植前与正常组比较,细胞移植组、对照组小鼠逃避潜伏期差异均有显著性意义(P<0.05);移植后2周细胞移植组小鼠逃避潜伏期与正常组基本相似(P>0.05),但明显短于对照组(P<0.05)。空间探索试验中,移植前后小鼠穿越平台次数及其在平台象限的时间3组间比较差异均无显著性意义(P>0.05)。移植后1个月,正常组未见或仅见极少量的β-淀粉样蛋白沉积,细胞移植组淀粉样蛋白的沉积明显少于对照组。结论:人羊膜间充质干细胞尾静脉移植能促进阿尔茨海默病转基因小鼠空间定位及学习记忆能力的提高,且减少小鼠脑内β-淀粉样蛋白的沉积。     

APP/PS1转基因AD小鼠脑内淀粉样斑块与铁沉积的磁敏感成像研究

目的 应用三维增强磁敏感成像(3D-enhanced susceptibility weighted angiography,ESWAN)技术检测APPswe/PSEN1de9基因突变型小鼠(购自美国Jackson Laboratory,简称为APP/PS1转基因小鼠)脑不同部位的平均相位值(mean phase value,MPV),并探讨其与铁蛋白及淀粉样斑块分布的相关性。材料与方法 APP/PS1转基因小鼠及野生型对照组小鼠行T1WI、T2WI和ESWAN序列扫描,测量小鼠脑皮质区、海马区及丘脑区MPV。对鼠脑组织进行铁蛋白和Aβ蛋白的免疫组化染色,并分别计数感兴趣区内染色斑块数量和面积所占百分比。对感兴趣区MPV与斑块数量、斑块面积百分比采用Pearson检验进行相关分析。结果 APP/PS1转基因小鼠与对照组小鼠皮质区(t=-2.201,P=0.045)和海马区(t=-2.524,P=0.024)的MPV值差异均具有统计学意义。APP/PS1转基因小鼠与对照组小鼠丘脑区的MPV差异不具有统计学意义(t=-2.094,P=0.055)。APP/PS1转基因小鼠每组中的MPV与铁蛋白斑块数量与范围分别具有显著相关性(P0.05);淀粉样斑块与铁蛋白的数量与范围分别具有相关性(P0.05)。结论 APP/PS1转基因小鼠脑内MPV与脑内铁沉积具有显著相关性。APP/PS1转基因小鼠脑内皮质区、海马区及丘脑区的铁沉积持续进行,并且与淀粉样斑块的形成具有相关性。     

黄连解毒汤对APP/PS1双转基因AD小鼠海马区病理形态学及脑内β-APP基因mRNA的影响

目的:探讨黄连解毒汤(HLJDD)对APP/PS1双转基因阿尔茨海默病(AD)模型小鼠海马CA1区的病理形态学及脑内β-淀粉样前体蛋白(β-APP)基因mRNA的影响。方法:选用3月龄的APP/PS1双转基因AD小鼠模型50只,随机分为对照组、安理申组、HLJDD大剂量组、HLJDD中剂量组、HLJDD小剂量组各10只,分别给予安理申或不同剂量HLJDD灌胃治疗6个月,对照组不予治疗,记录并比较各组死亡率;分别于6月龄和10月龄时采用HE染色观察各组海马CA1区神经细胞形态,采用改良甲醇刚果红染色观察各组老年斑形成情况;于末次灌胃治疗后,通过实时荧光定量PCR检测各组脑内β-APPmRNA水平。结果:各组小鼠死亡率差异无统计学意义;10月龄HLJDD各剂量组海马CA1区神经损伤情况较对照组明显减轻,老年斑数量少于对照组(P0.05);HLJDD各剂量组β-APPmRNA水平低于对照组(P0.05),其中以中剂量组水平降低最明显(P0.01)。结论:HLJDD能保护海马神经细胞。     

电针百会穴对APP/PS1双转基因小鼠学习记忆及脑源性神经营养因子表达的影响

目的观察电针百会穴对APP/PS1双转基因小鼠学习记忆功能及脑源性神经营养因子(BDNF)表达的影响,探讨电针治疗阿尔茨海默病的作用机制。方法将30只4月龄雌性APP/PS1双转基因小鼠随机分为模型组、百会组和非穴组,每组10只,另取10只同窝阴性野生小鼠为野生组。百会组电针百会穴,非穴组电针非穴,干预28 d,野生组和模型组不干预。采用Morris水迷宫实验观察小鼠学习记忆能力;免疫组化技术观察小鼠大脑皮质β-淀粉样蛋白的沉积;Western blotting和RT-PCR法检测小鼠皮质脑源性神经营养因子(BDNF)蛋白和基因的表达情况。结果与模型组相比,百会组小鼠逃避潜伏期缩短(P0.05),跨越平台次数明显增加(P0.01);β-淀粉样蛋白的沉积减少(P0.05);BDNF蛋白和基因的表达明显增多(P0.01)。非穴组与模型组相比无显著性差异(P0.05)。结论电针百会穴可改善APP/PS1双转基因小鼠的学习记忆功能,其作用机制可能与增加大脑皮质BDNF蛋白和基因的表达,降低β-淀粉样蛋白的沉积有关。     

淀粉样前体蛋白转基因鼠对β-淀粉样蛋白的免疫活性降低:与早老性痴呆致病机制和治疗的关系

阿尔兹海默病(alzheimer's disease,AD)与β-淀粉样蛋白(amyloid β-protein,Aβ)在脑组织中的积聚有关。β-淀粉样蛋白前体(APP)基因和早老素(PS1和PS2)基因的突变均导致Aβ产生和积累,通常Aβ积累出现于家族性AD患者和唐氏综合征病人体内。所以,Aβ可能在遗传性和偶发性AD形成过程中起着前提和核心作用。以转基因鼠为对象的研究表明有,     

APP/PS1小鼠不同时期灰质体积变化与Aβ沉积和学习记忆功能的相关性分析

目的:分析阿尔兹海默病(AD)双转基因APP/PS1模型小鼠大脑灰质体积变化与其学习记忆能力和相关脑区β-淀粉样蛋白(Aβ)沉积的相关性,以期为探明AD动物模型的病理特征提供可靠的行为学和影像学依据。方法:共纳入野生组(WT)和APP/PS1组2组小鼠,每组36只。WT组小鼠选用C57-BL/6小鼠,APP/PS1组选用APP/PS1双转基因小鼠,2组均分别由2、6、12不同月龄小鼠组成,每组每个月龄小鼠12只。通过Morris水迷宫测试(定位航行实验和空间探索实验)检测2组小鼠空间记忆学习能力和空间记忆提取能力;采用磁共振T2加权成像(MRI T2WI)扫描检测小鼠大脑双侧内嗅皮质和海马的灰质体积变化情况;分离和制备2组不同月龄小鼠大脑石蜡切片,采用Thioflavine-s(Th-s)荧光染色检测2组小鼠大脑双侧内嗅皮质和海马的Aβ沉积情况;采用Pearson相关分析法分析2组小鼠不同时期的灰质体积变化与其学习记忆能力和对应脑区Aβ沉积的相关性。结果:①Morris水迷宫测试结果:与WT组比较,APP/PS1组2月龄小鼠行为学结果无明显区别,差异无统计学意义(P0.05);APP/PS1组6、12月龄小鼠逃避潜伏期明显增加、穿越平台次数明显减少,差异具有统计学意义(P0.05)。②MRI T2WI扫描结果:与WT组比较,APP/PS1组2月龄小鼠灰质体积无明显变化,差异无统计学意义(P0.05);APP/PS1组6月龄小鼠内嗅皮质体积明显缩小,APP/PS1组12月龄小鼠内嗅皮质和海马体积均明显缩小,差异具有统计学意义(P0.05)。③Th-s荧光染色检测结果:WT组、APP/PS1组2月龄小鼠均未检测出Aβ沉积;与WT组比较,APP/PS1组小鼠6月龄内嗅皮质和海马可检测到少量Aβ沉积,而12月龄时内嗅皮质和海马则可检测出大量致密Aβ斑块,差异具有统计学意义(P0.05)。④相关性分析结果:APP/PS1组小鼠内嗅皮质和海马体积变化与逃避潜伏期呈明显负相关关系(r=-0.729,P0.001;r=-0.643,P0.001);APP/PS1组小鼠内嗅皮质和海马体积变化与穿越平台次数呈明显正相关关系(r=0.705,P0.001;r=0.719,P0.001);APP/PS1组小鼠内嗅皮质和海马体积变化与Aβ沉积呈明显负相关关系(r=-0.865,P0.001;r=-0.885,P0.001)。结论:APP/PS1转基因小鼠可能于6月龄时就发生学习记忆功能障碍,且随时间渐进性加重;这种学习记忆功能障碍可能与大脑内嗅皮质和海马体积萎缩有关;而Aβ过度沉积可能是导致大脑灰质体积萎缩的因素之一。     

电针对APP/PS1小鼠学习记忆及β位淀粉样前体蛋白裂解酶1表达的影响

目的探讨电针治疗阿尔茨海默病的效果和作用机制。方法雄性8月龄APP/PS1小鼠24只随机分为模型组、电针组和非穴组,各8只。8只野生型小鼠为野生组。电针组电针百会、神庭,非穴组电针双侧肋下非穴区,野生组和模型组予相同抓取和固定,共28 d。干预后,Morris水迷宫测试小鼠学习记忆能力,分别采用免疫荧光和免疫组化技术检测小鼠大脑皮质β淀粉样蛋白(Aβ)和β位淀粉样前体蛋白裂解酶1 (BACE1)含量,RT-PCR检测BACE1 m RNA水平。结果与模型组比较,电针组小鼠Morris水迷宫逃避潜伏期显著下降(P 0.001),穿越平台次数显著增加(P 0.001),BACE1表达和Aβ水平显著减少(P 0.001)。结论电针百会、神庭可能通过抑制APP/PS1小鼠大脑皮质BACE1表达,降低Aβ水平,改善小鼠学习记忆能力。     

沂蒙山区3年32例阿尔茨海默病患者淀粉样β蛋白及其前体基因表达量的相关性分析

背景:淀粉样β蛋白沉积是引致阿尔茨海默病的重要原因,淀粉样β蛋白前体基因过度表达或某部分发生突变,可能与阿尔茨海默病的发生有关。目的:观察沂蒙山区阿尔茨海默病与淀粉样β蛋白及其前体基因的关系。设计:病例-对照分析。单位:临沂市人民医院神经内科。对象:选择临沂医专附属临沂市人民医院神经内科2001-01/2003-12住院阿尔茨海默病患者32例,以性别、年龄相当的非颅脑损伤的外科手术老年人30例为对照组(简易精神状态量表评分>27分)。方法:应用ELISA法检测脑脊液淀粉样β蛋白水平,应用荧光定量聚合酶链反应技术检测淀粉样β蛋白前体基因表达量。结果:62例全部进入结果分析。①阿尔茨海默病组脑脊液淀粉样β蛋白水平明显高于对照组[(13.63±9.34),(6.75±5.37)μg/L,t=3.354,P<0.01]。②阿尔茨海默病组脑脊液淀粉样β蛋白前体表达量明显高于对照组[(1624±298),(1275±269)拷贝/μL,t=4.42,P<0.01]。③阿尔茨海默病组淀粉样β蛋白水平与淀粉样β蛋白前体基因表达量呈正相关(r=0.44,P<0.01)。结论:沂蒙山区阿尔茨海默病患者脑脊液中淀粉样β蛋白及其前体基因表达增高,且淀粉样β蛋白水平越高,其前体基因表达越高,痴呆程度越重。提示阿尔茨海默病与淀粉样β蛋白、淀粉样β蛋白前体密切相关。但淀粉样β蛋白前体表达量在阿尔茨海默病诊断中不具特异性。     

Pharmacokinetic analysis of the blood-brain barrier transport of 125I-amyloid beta protein 40 in wild-type and Alzheimer's disease transgenic mice (APP,PS1) and its implications for amyloid plaque formation

Amyloid plaques are formed in the extracellular space of Alzheimer's disease (AD) brain due to the accumulation of amyloid beta (Abeta) proteins such as Abeta40. The relationship between Abeta40 pharmacokinetics and its accumulation within and clearance from the brain in both wild-type (WT) and AD transgenic mice (APP,PS1) was studied to understand the mechanism of amyloid plaque formation and the potential use of Abeta40 as a probe to target and detect amyloid plaques. In both WT and APP,PS1 mice, the (125)I-Abeta40 tracer exhibited biexponential disposition in plasma with very short first and second phase half-lives. The (125)I-Abeta40 was significantly metabolized in the liver kidney > spleen. Coadministration of exogenous Abeta40 inhibited the plasma clearance and the uptake of (125)I-Abeta40 at the blood-brain barrier (BBB) in WT animals but did not affect its elimination from the brain. The (125)I-Abeta40 was shown to be metabolized within and effluxed from the brain parenchyma. The rate of efflux from APP,PS1 brain slices was substantially lower compared with WT brain slices. Since the Abeta40 receptor at the BBB can be easily saturated, the blood-to-brain transport of Abeta40 is less likely to be a primary contributor to the amyloid plaque formation in APP,PS1 mice. The decreased elimination of Abeta40 from the brain is most likely responsible for the amyloid plaque formation in the brain of APP,PS1 mice. Furthermore, inadequate targeting of Abeta40 to amyloid plaques, despite its high BBB permeability, is due to the saturability of Abeta40 transporter at the BBB and its metabolism and efflux from the brain.     

喹硫平对阿尔茨海默小鼠淀粉样β蛋白42表达的影响及机制研究

目的探索喹硫平对阿尔茨海默小鼠淀粉样β蛋白42(Aβ42)的表达的影响及其机制。方法C57BL/6小鼠20只作为对照组,APP/PS1小鼠40只分为APP/PS1组、APP/PS1+喹硫平组,各20只。APP/PS1+喹硫平组小鼠通过腹腔给予喹硫平溶液2.5 mg/(kg·d),连续给药2个月,对照组和APP/PS1组小鼠每天腹腔给予等量双蒸水。通过新事物识别实验检测三组小鼠的记忆功能;通过Western blot检测三组小鼠海马脑区Aβ42蛋白、核因子-κB(NF-κB)p65蛋白的表达水平;通过酶联免疫吸附(ELISA)检测海马脑区白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α)的表达水平,免疫荧光染色计算小胶质细胞数量。结果与对照组比较,APP/PS1小鼠对新事物的探索时间显著降低(P<0.05),小胶质细胞明显激活,小胶质细胞的数量显著增加(P<0.05),IL-10和TNF-α的表达水平均显著增加(P<0.05),Aβ42、NF-κB p65蛋白表达水平显著增加(P<0.05);与APP/PS1组比较,APP/PS1+喹硫平组小鼠的探索时间显著延长(P<0.05),小胶质细胞数量显著减少(P<0.05),IL-10和TNF-α表达水平均显著降低(P<0.05),Aβ42、NF-κB p65蛋白表达水平显著降低(P<0.05)。结论喹硫平能降低Aβ42蛋白的产生,促进AD小鼠的记忆功能恢复,其发生机制可能与小胶质细胞激活和NF-κB信号通路活化相关。     

Pharmacokinetics and amyloid plaque targeting ability of a novel peptide-based magnetic resonance contrast agent in wild-type and Alzheimer's disease transgenic mice

A novel magnetic resonance (MR) imaging contrast agent based on a derivative of human amyloid beta (Abeta) peptide, Gd[N-4ab/Q-4ab]Abeta 30, was previously shown to cross the blood-brain barrier (BBB) and bind to amyloid plaques in Alzheimer's disease (AD) transgenic mouse (APP/PS1) brain. We now report extensive plasma and brain pharmacokinetics of this contrast agent in wild-type (WT) and in APP/PS1 mice along with a quantitative summary of various physiological factors that govern its efficacy. Upon i.v. bolus administration, (125)I-Gd[N-4ab/Q-4ab]Abeta 30 was rapidly eliminated from the plasma following a three-exponential disposition, which is saturable at higher concentrations. Nevertheless, the contrast agent exhibited rapid and nonsaturable absorption at the BBB. The brain pharmacokinetic profile of (125)I-Gd[N-4ab/Q-4ab]Abeta 30 showed a rapid absorption phase followed by a slower elimination phase. No significant differences were observed in the plasma or brain kinetics of WT and APP/PS1 animals. Emulsion autoradiography studies conducted on WT and APP/PS1 mouse brain after an i.v. bolus administration of (125)I-Gd[N-4ab/Q-4ab]Abeta 30 in vivo confirmed the brain pharmacokinetic data and also demonstrated the preferential localization of the contrast agent on the plaques for an extended period of time. These attributes of the contrast agent are extremely useful in providing an excellent signal/noise ratio during longer MR scans, which may be essential for obtaining a high resolution image. In conclusion, this study documents the successful plaque targeting of Gd[N-4ab/Q-4ab]Abeta 30 and provides crucial pharmacokinetic information to determine the dose, mode of administration, and scan times for future in vivo MR imaging of amyloid plaques in AD transgenic mice.     

转基因阿尔茨海默病小鼠脑内炎症反应的诱发因素研究

目的 观察炎症反应在转基因阿尔茨海默病(AD)小鼠脑组织中的变化,探讨AD脑内炎症反应的诱发因素.方法 选用3、12个月龄转人β-淀粉样前体蛋白/早老素-1(APP/PS1)基因AD小鼠及正常野生型(WT)小鼠,分别应用免疫组织化学法和ELISA法观察脑内淀粉样斑块、炎性因子[白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)α、前列腺素(PGE)2]的变化.结果 3个月龄APP/PS1基因AD小鼠脑组织中无淀粉样斑块沉积,未发现激活的星型胶质细胞和小胶质细胞,炎性因子IL-1β、IL-6、TNFα、PGE2的含量与WT小鼠差异无统计学意义(P均＞0.05).12个月龄转APP/PS1基因AD小鼠脑组织中有大量淀粉样斑块沉积,并伴有大量激活的星型胶质细胞和小胶质细胞,炎性因子IL-1β、IL-6、TNFα、PGE2的含量[分别为(56.02±9.04)、(8.66±0.83)、(97.48±26.58)、(72.18±21.01)ng/g]较WT小鼠[分别为(29.81±6.03)、(7.73±0.74)、(61.98±11.11)、(37.23±10.96)ng/g]及3个月龄转APP/PS1基因AD小鼠[分别为(30.05±3.53)、(7.43±1.17)、(59.34±10.47)、(42.56±5.93)ng/g]显著增加(P＜0.05或P＜0.01).结论 在淀粉样斑块形成之前,转APP/PS1基因AD小鼠脑组织中无明确的炎症反应;而淀粉样斑块沉积之后,脑组织中有显著的炎症反应;AD脑内炎症反应与淀粉样斑块形成密切相关,淀粉样蛋白(Aβ)沉积是导致脑内炎症反应的直接诱发因素.

Abstract：

Objective To observe the changes of cerebral inflammation-related markers in brain of a transgenic mouse model of Alzheimer's disease (AD) ,and to determine the causative factor to the development of cerebral inflammation in AD. Methods 3- and 12-month-old β-amyloid protein precursor ( APP)/presenilin (PSI) transgenic mice and age-matched wild-type mice (WT) were used in the study. The changes of amyloid plaques, inflammatory factors ( interleukin 1β ( IL-1β ); interleukin 6( IL-6 ); tumor necrosis factor α (TNFα) ;prostaglandin E2 (PGE2)) in the brains among these mice were measured by immunohistochemistry and ELISA. Results Immunohistochemical analysis revealed that no amyloid plaques and activated astrocytes as well as microglia were observed in the 3-month-old APP/PS1 mice. There were no significant differences in the levels of inflammatory factors (IL-1β, IL-6 ,TNFα,and PGE2) between the 3-month-old APP/PS1 and WT mice ( Ps ＞ 0. 05 ). However, abundant amyloid plaques accompanied by a remarkable increase of activated astrocytes and microglia were found in the brain of the 12-month-old APP/PS1 mice. The levels of inflammatory factors (IL-1β,IL-6,TNFα, and PGE2 ) were significantly increased in the 12-month-old APP/PS1 mice ([56. 02 ±9. 04] ng/g, [8. 66 ±0.83] ng/g, [97.48 ±26.58] ng/g, [72. 18 ±21.01] ng/g) than in the WT mice ([29. 18 ± 6. 03] ng/g, [7. 73 ± 0. 74] ng/g, [61.98 ±11.11] ng/g, [37. 23 ± 10. 96] ng/g) and the 3-month-old APP/PS1 mice ( [30. 05 ± 3.53] ng/g, [7.43 ± 1.17] ng/g, [59.34 ± 10. 07] ng/g, [42. 56 ±5.93] ng/g) (P＜0.05,or P＜0.01,respectively). Conclusion This study demonstrates that the APP/PS1mice did not show cerebral inflammation before the appearance of amyloid plaques, and exhibited remarkable inflammation after amyloid plaque deposition. These findings suggest that the induction of cerebral inflammation is tightly associated with amyloid plaque formation, and deposition of amyloid-beta protein (Aβ) may be the direct causative factor to the development of cerebral inflammation in AD.     

Detection of amyloid plaques targeted by USPIO-Aβ1-42 in Alzheimer's disease transgenic mice using magnetic resonance microimaging

Amyloid plaques are one of the pathological hallmarks of Alzheimer's disease (AD). The visualization of amyloid plaques in the brain is important to monitor AD progression and to evaluate the efficacy of therapeutic interventions. Our group has developed several contrast agents to detect amyloid plaques in vivo using magnetic resonance microimaging (μMRI) in AD transgenic mice, where we used intra-carotid mannitol to enhance blood-brain barrier (BBB) permeability. In the present study, we used ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, chemically coupled with Aβ1-42 peptide to detect amyloid deposition along with mannitol for in vivo μMRI by femoral intravenous injection. A 3D gradient multi-echo sequence was used for imaging with a 100μm isotropic resolution. The amyloid plaques detected by T2*-weighted μMRI were confirmed with matched histological sections. Furthermore, two different quantitative analyses were used. The region of interest-based quantitative measurement of T2* values showed contrast-injected APP/PS1 mice had significantly reduced T2* values compared to wild-type mice. In addition, the scans were examined with voxel-based morphometry (VBM) using statistical parametric mapping (SPM) for comparison of contrast-injected AD transgenic and wild-type mice. The regional differences seen in VBM comparing USPIO-Aβ1-42 injected APP/PS1 and wild-type mice correlated with the amyloid plaque distribution histologically, contrasting with no differences between the two groups of mice without contrast agent injection in regions of the brain with amyloid deposition. Our results demonstrated that both approaches were able to identify the differences between AD transgenic mice and wild-type mice, after injected with USPIO-Aβ1-42. The feasibility of using less invasive intravenous femoral injections for amyloid plaque detection in AD transgenic mice facilitates using this method for longitudinal studies in the pathogenesis of AD.     

阿尔茨海默病转基因小鼠早期记忆功能障碍与氧化应激损伤关系的实验研究

目的 观察转APP/PS1基因阿尔茨海默病(AD)小鼠(APP/PS1小鼠)早期空间学习记忆功能及相关氧化应激反应指标的变化,并探讨它们之间的相关性.方法 应用Morris水迷宫评定APP/PS1小鼠及相应野生型(WT)小鼠的空间学习记忆功能,采用ELISA方法检测脑组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活性以及丙二醛(MDA)和蛋白质羰基的含量,并进行相关性分析.结果 2组小鼠的空间学习能力无明显差异(P＞0.05),而APP/PS1小鼠在目标象限(定位航行实验中平台所置第二象限)中航行时间占总时间的百分比(29.02±4.27)%较WT小鼠(47.39±6.01)%显著下降(t=0.000,P＜0.05),APP/PS1小鼠在目标象限中航行路程占总路程的百分比(28.85±3.77)%较WT小鼠(46.70±5.60)%也显著下降(t=0.000,P＜0.05),提示APP/PS1小鼠的空间记忆功能较WT小鼠显著下降.2组小鼠脑组织中的MDA含量、SOD和GSH-PX活性均无明显差异(P均＞0.05),而APP/PS1小鼠脑组织中的蛋白质羰基含量(2.67±0.19)较WT小鼠(2.38±0.15)显著增加(t=0.0088,P＜0.05).相关性分析表明:APP/PS1小鼠蛋白质羰基含量与目标象限航行时间百分比呈显著负性相关(r=-0.639,P＜0.05),APP/PS1小鼠蛋白质羰基含量与目标象限航行路程百分比呈显著负性相关(r=-0.636,P＜0.05).结论 APP/PS1小鼠早期空间记忆功能损害与脑组织中的蛋白质羰基含量升高呈负性相关,提示氧化应激导致的蛋白质羰基化在AD早期记忆损害发病过程中具有重要作用.

Abstract：

Objective To investigate the spatial learning and memory ability,the changes of indicators of oxidative stress,and their relationship in transgenic APP/PS1 mouse model of Alzheimer's disease(APP/PS1 mice). Methods The spatial learning and memory ability were assessed by Morris water maze test,and the activity or content of SOD, GSH-PX, MDA, and protein carbonyl in brain tissues were measured by ELISA in the APP/PS1 and wild type (WT) mice. Furthermore, the relationship between the learning and memory performances and the indicators of oxidative stress was examined. Results No significant difference in the spatial learning was observed between the APP/PS1 and WT mice (P ＜0. 05). The spatial memory which was measured as the percentage of time traveling in the targeted quadrant to the total traveling time was significantlydeclined in the APP/PS1 mice(29. 02 ± 4. 27) % as compared with the WT mice(47. 39 ± 6. 01) %(t =0. 000 ,P ＜0. 05). The percentage of length of traveling in the targeted quadrant to the total length traveled was significantly lower in the APP/PS1 mice(28. 85 ±3.77)% compared with the WT mice(46. 70 ±5.60)% (t =0. 000,P ＜0. 05). These findings indicated that the spatial learning and memory ability of APP/PS1 mice was significantly decreased compared to WT mice. There was no significant difference in activity or content of SOD,GSH-PX,and MDA in brain tissues between the APP/PS1 and WT mice (P ＜ 0. 05), while the content of protein carbonyl was significantly elevated in the APP/PS1 mice (2. 67 ±0. 19) than in the WT mice (2. 38 ±0. 15)(t = 0. 008, P ＜ 0. 05). Correlation analysis revealed that the elevated protein carbonyl was negatively correlated with the percentage of length traveled in the targeted quadrant(r = - 0. 639, P ＜ 0. 05) and the percentage of time traveled in the targeted quadrant(r = - 0. 636 ,P ＜ 0. 05). Conclusion The spatial memory impairment was negatively correlated with the elevated protein carbonyl in the APP/PS1 mice, suggesting that protein carbonylation caused by oxidative stress might play an important role in the development of memory impairment in the early stage of Alzheimer's disease.     

In vivo beta-secretase 1 inhibition leads to brain Abeta lowering and increased alpha-secretase processing of amyloid precursor protein without effect on neuregulin-1

beta-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid beta peptide Abeta42, the putative neurotoxic species in Alzheimer's disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC(50) approximately 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose- and time-dependent lowering of brain Abeta40 and Abeta42. BACE1 inhibition also led to a robust brain secreted (s)APPbeta lowering that was accompanied by an increase in brain sAPPalpha levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (-/-) mice, in agreement with previous studies, this effect was also observed in 15-day-old heterozygous (+/-) mice, but it was not evident in 30-day-old and 2-year-old BACE1 KO (-/-) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant Abeta lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major beta-site cleavage enzyme for APP and that its inhibition can lower brain Abeta and redirect APP processing via the potentially nonamyloidogenic alpha-secretase pathway, without significantly altering NRG-1 processing.     

Memantine improves spatial learning in a transgenic mouse model of Alzheimer's disease

Memantine, a low- to moderate-affinity uncompetitive N-methyl-D-aspartate receptor antagonist, has been shown to improve learning and memory in several pharmacological models of Alzheimer's disease (AD). In the present study, the effect of memantine on locomotor activity, social behavior, and spatial learning was assessed in a transgenic mouse model of AD. Eight-month-old male C57BL/6J mice carrying mutated human APP and PS1 genes (APP/PS1) and their nontransgenic (NT) litter mates were administered a therapeutic dose of memantine (30 mg/kg/day p.o.) for 2 to 3 weeks. At this age, APP/PS1 mice show elevated levels of beta-amyloid peptides in several brain regions. APP/PS1 mice exhibited less exploratory rearing and increased aggressive behavior compared with NT mice. In the water maze test for spatial learning, APP/PS1 mice had longer escape latencies to both hidden and visible platforms, but they did not differ from NT mice in their swimming speed. Memantine significantly improved the acquisition of the water maze in APP/PS1 mice without affecting swimming speed. Memantine did not affect either locomotor activity or aggressive behavior in either genotype. These data indicate that memantine improves hippocampus-based spatial learning in a transgenic mouse model of AD without producing nonspecific effects on locomotion/exploratory activity.     

Deficits in axonal transport in hippocampal-based circuitry and the visual pathway in APP knock-out animals witnessed by manganese enhanced MRI

Mounting evidence implicates axonal transport defects, typified by the presence of axonal varicosities with aberrant accumulations of cargo, as an early event in Alzheimer's disease (AD) pathogenesis. Work identifying amyloid precursor protein (APP) as a vesicular motor receptor for anterograde axonal transport further implicates axonal transport in AD. Manganese-enhanced MRI (MEMRI) detects axonal transport dynamics in preclinical studies. Here we pursue an understanding of the role of APP in axonal transport in the central nervous system by applying MEMRI to hippocampal circuitry and to the visual pathway in living mice homozygous for either wild type or a deletion in the APP gene (n=12 for each genotype). Following intra-ocular or stereotaxic hippocampal injection, we performed time-lapse MRI to detect Mn(2+) transport. Three dimensional whole brain datasets were compared on a voxel-wise basis using within-group pair-wise analysis. Quantification of transport to structures connected to injection sites via axonal fiber tracts was also performed. Histology confirmed consistent placement of hippocampal injections and no observable difference in glial-response to the injections. APP-/- mice had significantly reduced transport from the hippocampus to the septal nuclei and amygdala after 7h and reduced transport to the contralateral hippocampus after 25 h; axonal transport deficits in the APP-/- animals were also identified in the visual pathway. These data support a system-wide role for APP in axonal transport within the central nervous system and demonstrate the power of MEMRI for assessing neuronal circuitry involved in memory and learning.     

CXCR3 promotes plaque formation and behavioral deficits in an Alzheimer’s disease model

Chemokines are important modulators of neuroinflammation and neurodegeneration. In the brains of Alzheimer’s disease (AD) patients and in AD animal models, the chemokine CXCL10 is found in high concentrations, suggesting a pathogenic role for this chemokine and its receptor, CXCR3. Recent studies aimed at addressing the role of CXCR3 in neurological diseases indicate potent, but diverse, functions for CXCR3. Here, we examined the impact of CXCR3 in the amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model of AD. We found that, compared with control APP/PSI animals, plaque burden and Aβ levels were strongly reduced in CXCR3-deficient APP/PS1 mice. Analysis of microglial phagocytosis in vitro and in vivo demonstrated that CXCR3 deficiency increased the microglial uptake of Aβ. Application of a CXCR3 antagonist increased microglial Aβ phagocytosis, which was associated with reduced TNF-α secretion. Moreover, in CXCR3-deficient APP/PS1 mice, microglia exhibited morphological activation and reduced plaque association, and brain tissue from APP/PS1 animals lacking CXCR3 had reduced concentrations of proinflammatory cytokines compared with controls. Further, loss of CXCR3 attenuated the behavioral deficits observed in APP/PS1 mice. Together, our data indicate that CXCR3 signaling mediates development of AD-like pathology in APP/PS1 mice and suggest that CXCR3 has potential as a therapeutic target for AD.