APP/PS1阿尔茨海默病转基因小鼠白质异常特点及其与脑内淀粉样病变之间关系的研究
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摘要
研究背景与目的
阿尔茨海默病(AD)是一种高发病率的神经系统变性疾病，临床上以逐渐进展且不可逆转的行为和认知障碍为特点。在AD进展过程中脑内病理学改变主要涉及2个方面：细胞外淀粉样蛋白β(Aβ)沉积形成的老年斑和细胞内tau蛋白积聚形成的神经纤维缠绕并最终促进细胞骨架的破坏、分解。目前AD的研究大部分是以一个重要的生理病理学假设所引导的，即所谓的“淀粉样蛋白瀑布学说”，该假说认为Aβ在脑内的沉积在AD疾病发生发展过程中扮演了一个最主要的角色，Aβ的沉积决定了脑内其他病理学改变的发生(如：突触和神经元的死亡)，并且最终导致了痴呆的临床阶段。其中最支持淀粉样蛋白瀑布学说的依据来源于对早发性家族性AD疾病的研究，早发性家族性AD与不同的基因突变有关：淀粉样蛋白前体蛋白基因(APP)和早老素1&2基因(PS1和PS2)，他们均与Aβ的生物合成有关。这些基因的机能障碍被认为导致APP代谢的异常从而最终导致Aβ过多产生。在表达突变的一个或几个上述基因的转基因小鼠中，我们观察到Aβ产生的增加和脑实质中淀粉样斑块的沉积，这些小鼠随后还出现了神经病理学的改变以及模拟AD表型的行为学的异常改变。
到目前为止，无论是在AD病人还是AD疾病动物模型中，脑内Aβ的沉积对临床痴呆症状的确切影响还没有找到直接的证据。临床病理学相关分析已经得到了一些有希望的结论：形成斑块之前的可溶性Aβ集合现在被认为是最具有毒性的，相反，积聚的不溶性Aβ集合致病性较弱。除此之外，Aβ的沉积和脑功能障碍的关系可能是间接的，他们被中间的神经病理学改变所调控。例如，AD病人脑内存在白质的异常，推测与脑-血管的异常相关，在模拟脑内淀粉样改变的转基因小鼠脑内，我们不仅能通过传统的神经病理学检测，而且能在体通过高新技术，如扩散增强成像技术，观察到该转基因小鼠脑内也存在白质的损害。最近有学者提出：白质缺陷可能是AD很重要的诊断性生物标记物。白质改变所带来的后果是显而易见的：神经网络的断开，随之而来纤维的丢失，最终导致皮层机能的紊乱；中枢神经系统髓鞘的崩解通过改变动作电位传播以及增加脑能量的输出，对神经元活动有致命的影响。
白质异常可能反映了轴突的丢失，也可能是脱髓鞘，并且确切地区分这两种过程十分困难。已有报道表明，AD病人不仅有轴突密度的下降，同时也存在髓鞘的崩解。
从AD动物模型的实验研究，可以明确地推出脑内Aβ的沉积可导致轴突的病理改变，这一点为联系脑内术期淀粉样改变和行为／认知障碍之间架起了一座桥梁。在本研究中，我们利用APP／PS1双转基因小鼠进一步评价了AD转基因小鼠白质的完整性。胼胝体和前联合在AD病人中有很大的改变，因此我们选择这两个区域作为白质的代表区域，通过神经纤维丝免疫组织化学染色分析了该区的轴突密度，通过氯化金染色评价了髓鞘的完整性；并且我们观察了APP／PS1小鼠白质的年龄相关的异常改变特点，进一步评价了这些异常改变和脑内淀粉样改变之间的关系。
方法
[1]对Schmued氯化金髓鞘染色进行了一些改动，并且采用该方法对不同年龄组大鼠的髓鞘染色进行定量分析，以及通过对不同表型小鼠的髓鞘染色进行定性分析。
[2]相邻的APP／PS1小鼠脑组织片分别采用氯化金髓鞘染色和抗神经微丝轴突染色，取胼胝体和前联合作为白质染色的代表区域，分别测量了代表区域平均吸光度值ROD，定量分析比较了该区域不同表型以及不同年龄小鼠之间髓鞘密度和轴突密度改变情况。
[3]细胞外淀粉样蛋白沉积采用标准的刚果红染色观察。采用基于计算机阈值方法，利用软件自动挑选有斑块的地方，根据Delesse原则计算刚果红染色区域占脑组织总面积的百分比，从而计算出淀粉样蛋白的含量。全脑、皮层运动区以及两个白质代表区域的淀粉样蛋白含量分别通过上述方法计算得出；然后分别对全脑和两个白质代表区域的淀粉样蛋白含量与该区域髓鞘密度、轴突密度以及代表区域面积大小进行了相关性分析。
[4]细胞内淀粉样蛋白通过4G8抗体进行染色，对阳性染色进行了半定量分析，其结果与前脑胼胝体面积大小进行相关性分析。
[5]神经元退行性变的评价采用Fluoro-Jade B染料染色方法，其结果与髓鞘以及轴突密度改变进行相关性分析。
结果
[1]新生7d大鼠髓鞘化染色阴性，21d大鼠的髓鞘化程度比14d大鼠的髓鞘化程度明显增高。APP／PS1组小鼠可观察到病理性髓鞘染色，定量分析显示其目标区域相对光密度值(ROD)低于PS1组，APP／PS1组存在明显的脱髓鞘改变。
[2]在脑组织冠状位切片上，氯化金髓鞘染色可以比较精确地确定胼胝体的轮廓，特别是将胼胝体与其周围相邻的白质纤维分开(例如：扣带回、背侧穹窿、背侧海马联合纤维)变得十分便捷；同时，胼胝体侧边的边缘和外囊的界限通过纤维不同方向走形达到区别。前联合在脑组织冠状位切片上很容易识别。
[3]APP／PS1小鼠纤维束体积的异常改变
在2月龄小鼠组，APP／PS1小鼠的前联合大小和PS1组小鼠的前联合大小是相当的(t(13)=-0.58，p＞0.05)；相反，APP／PS1组小鼠胼胝体大小比PS1组显著地减少了(t(13)=3.501，p＜0.005)。胼胝体不同亚区分析显示，胼胝体的减小主要集中在嘴部(t(13)=3.743，p＜0.005)。随着年龄的增长，PS1对照组白质体积有显著地增加(胼胝体：t(12)=3.858，p＜0.005；前联合：t(12)=4.275，p＜0.005)。然而，在APP／PS1转基因小鼠这种现象并不存在。胼胝体大小在2月龄和24月龄之间是相当的(t(12)=1.850，p＞0.05)，并且前联合的表面积随着年龄的增长不断地萎缩(t(12)=2.284，p＜0.05)。老年组APP／PS1小鼠前联合(t(11)=6.388，p＜0.0001)和胼胝体(胼胝体总：t(11)=4.653，p＜0.001；胼胝体嘴部：t(11)=5.404，p＜0.0005)比老年组PS1组小鼠前联合和胼胝体有显著地缩小。但是，在老年组胼胝体后部，PS1组和APP／PS1组之间没有区别(t(11)=1.492，p＞0.05)。
[4]老年组APP／PS1小鼠轴突丢失突出：在2月龄小鼠，2组小鼠的轴突密度都是相当的(p＞0.35)。随着年龄的增长，无论是在APP／PS1组还是PS1组，神经微丝染色都有严重的下降(p＜0.0001)，从胼胝体和前联合的统计学数据可以确定。然而，年龄相关的神经纤维染色的下降在老年组APP／PS1比老年组PS1组更加突出(p＜0.0001)。
[5]老年组APP／PS1小鼠异常的髓鞘染色定量分析：在2月龄组小鼠，氯化金染色的ROD分析显示PS1组和APP／PS1组的髓鞘密度是相当的(胼胝体：t(13)=0.318，p＞0.05；前联合：t(13)=1.277，p＞0.05)。
前联合的髓鞘化程度并没有随着年龄的影响而有所下降(PS1：t(12)=1.292，p＞0.05；APPxPS1：t(12)=0.556，p＞0.05)，因此在24月龄PS1组和APP／PS1组的前联合处髓鞘化密度是相当的(t(11)=0.679，p＞0.05)。然而，24月龄PS1小鼠胼胝体的髓鞘化程度比2月龄PS1有增高(t(12)=2.823，p＜0.05)，并且值得注意的是，随着年龄的增长，髓鞘化的程度主要发生在胼胝体的嘴部(t(12)=4.171，p＜0.005)，而在胼胝体后部并不存在这种情况(t(12)=0.461，p＞0.05)。和PS1小鼠相反，年龄相关的胼胝体不断髓鞘化过程并不存在于APP／PS1小鼠组(t(12)=0.7，p＞0.05)，因此24月龄APP／PS1小鼠的髓鞘化染色比24月龄PS1小鼠有明显的下降(总胼胝体：t(11)=3.332，p＜0.01)；并且这种髓鞘染色的下降主要位于胼胝体的嘴部(t(11)=3.512，p＜0.005)。在胼胝体后部PS1组和APP／PS1组之间没有区别(t(11)=1.9，p＞0.05)。
[6]光学显微镜下，老年组APP／PS1小鼠异常的髓鞘染色定性分析：髓鞘染色的定性分析检测，在老年组APP／PS1小鼠，在大的髓鞘化纤维束，没有明显的髓鞘崩解；但是和PS1对照组小鼠比较，APP／PS1小鼠的新皮层和海马区可以看到断裂的髓鞘碎片，髓鞘物质以小的扭曲的片段存在，并且带有“静脉曲张样改变”。这些形态学的异常(在年轻组APP／PS1小鼠组看不到)不仅出现在Aβ聚集的边缘，在远离Aβ斑块的脑组织实质中也可以看到。
[7]全脑及皮层兴趣区Aβ的含量和纤维束异常之间的关联分析在老年组APP／PS1组小鼠，定量测量了刚果红阳性的集聚物(前联合：Aβ平均含量=2.8％，最小值=1.6％，最大值=6％；胼胝体：Aβ平均含量=2.2％，最小值=1.6％，最大值=2.7％)。相关性分析表明，纤维束局部淀粉样蛋白含量和白质异常(包括轴突密度的下降和脱髓鞘)并不存在关联(所有的p＞0.111)。而且，全脑或皮层的淀粉样蛋白含量与胼胝体和前联合的大小之间也都没有关联(所有的p＞0.196)
[8]脑内皮质V层细胞内Aβ的含量的半定量测定和纤维束异常之间的关联分析在年轻组APP／PS1小鼠的前皮层，细胞内Aβ被半定量分析。和以往的观察一致，通过4G8抗体可以观察到阳性的染色。细胞内Aβ主要存在于皮层V。尽管细胞内Aβ水平在每个小鼠中半定量变化很大(平均值=7.8；最小值=4.5；最大值11.5)，但是相关性分析仍表明细胞内Aβ含量与轴突和髓鞘密度改变之间没有关联(所有p＞0.1119)。
[9]APP／PS1小鼠皮层变性神经元
采用Fluoro-Jade B染料观察APP／PS1小鼠神经元退行性变化。我们在研究动物中并未发现阳性的神经元染色。即使是在含有大量细胞内Aβ阳性皮层神经元中，我们也未发现退行性变化的神经元。仅仅在老年组APP／PS1小鼠，我们看能观察到淀粉样沉积的核心以及围绕在其周围退行性变化的神经突和反应性星形胶质细胞。
结论：
[1]改良的氯化金髓鞘染色快速、简单、敏感、稳定，可进行髓鞘化定性、定量分析。
[2]利用氯化金髓鞘染色，可以较精确地勾画胼胝体的轮廓并且测量其大小。为胼胝体大小的定量研究提供了一个简单而巧妙的方法。
[3]老年组APPxPS1前脑纤维束存在着严重萎缩，其发生不仅和轴突丢失有关，也和脱髓鞘有关。但APP／PS1小鼠严重的轴突丢失更加能解释纤维束的萎缩。
[4]无论是整个脑内还是局部脑细胞外淀粉样蛋白Aβ的含量，与老年组APP／PS1前脑纤维束的异常病理学改变，包括轴突的丢失和脱髓鞘导致的纤维束的萎缩，他们之间都不存在关联。即细胞外Aβ淀粉样蛋白并没有明确的致病性。
[5]细胞内β淀粉样蛋白的沉积，主要沉积皮层V，而该皮层细胞正好是胼胝体所对应的输入输出纤维的细胞体所在地，早期细胞内β淀粉样蛋白的沉积，随着年龄的增长，细胞会逐渐失去其功能，最终出现轴突丢失和脱髓鞘的病理学改变。
Alzheimer's disease(AD) is a high prevalence neurodegenerative disease accompanied bygradual and irreversible behavioral and cognitive impairments. Brain lesions observedduring the course of AD involve two main aspects: extracellular amyloid-beta(Aβ)deposition as senile plaques and intracellular tau accumulation forming neurofibrillarytangles and promoting cytoskeletal disorganization. Current research on AD is largelyguided by a dominant physiopathogenic hypothesis, the so-called amyloid cascade theory.Regularly commented on and amended, this model posits accumulation of Aβin thebrain, as a key primary event that determines the onset of other brain alterations(e.g.synaptic and neuronal death), finally leading to the clinical stage of dementia. Supportingthe amyloid cascade hypothesis, early-onset familial forms of AD are associated withmutations in different genes(Amyloid Precursor Protein(APP) and Presenilins 1&2,(PS1&2)) involved in the biosynthesis of Aβ. Dysfunction of these genes is logicallythought to compromise the normal catabolism of APP resulting in exaggerated Aβproduction. Increased Aβproduction and parenchymal amyloid plaques are indeeddescribed in transgenic mice overexpressing one or more of these mutated genes. Thesemice subsequently develop neuropathological alterations and behavioural impairmentsmimicking AD phenotype.
The exact impact of brain Aβaccumulation on clinical symptoms remains to-date difficultto decipher, both in AD patients and in animal models of the disease. Clinico-pathologicalcorrelative analyses have led to mitigated conclusions and it is now considered thatpre-plaques Aβassemblies are the most deleterious species while aggregated insolubledeposits have a reduced pathogenicity. In addition, the relationship between Aβaccumulation and brain dysfunction might be indirect and mediated by secondaryneuropathological alterations. For instance, white matter anomalies are described in ADpatients, presumably in association with cerebro-vascular impairments, and in transgenicmice modeling brain amyloidosis. They can be detected through conventionalpostmortem neuropathological examination, but also in vivo by means of dedicatedtechniques such as diffusion tensor imaging and it has recently been proposed that white matter defects are potent diagnostic biomarkers for AD. The functionalconsequences of altered white matter are obvious: Disconnection of neural networks occurfollowing fibres loss, leading to diaschesis and cortical disorganized activity. Also,disruption of myelin in the CNS white matter might have deleterious effects on neuronalcommunication by altering propagation of action potentials and increasing brain energyexpenditure.
Importantly, white matter anomalies might reflect either loss of fibers and/or demyelinationbut deciphering between the two processes could sometimes be hazardous. In particular, ithas been demonstrated that AD patients show concurrent decreased axonal densities andmyelin breakdown.
It is clear, especially from experimental studies in AD animal models, that axonal pathologycan be driven by Aβdeposition in the brain therefore possibly bridging the gapbetween amyloidosis and behavioural/cognitive impairments.
The aim of the present study was to further evaluate white matter integrity in a doubleAPPxPS1 mouse transgenic model with aggressive Aβ-related pathology. Twoaxonal bundles(corpus callosum and anterior commissure) that show significant alterationsin AD patients were selected and analyzed: axonal densities were quantified by meansof anti-neurofilament immunostainings and myelin integrity was evaluated byhistochemistry using a gold chloride staining that provides high contrast and spatialresolution. Age-dependent anomalies were detected in the white matter of APPxPS1mice and we further assessed the relationship between these alterations and Aβdeposition.
Methods
1) Three groups of new-born rats, each group contained 5 rats, which were 7 days、14days and 21 days old. The other 13 Alzheimer's disease transgenic rats, contained 5APP/PS1 mol/Lice and PS1 mol/Lice. Following decapitation the brains were extractedand treated. The brains were cut by the microtome. The brain tissue was stained byimproved gold chloride myelin staining. Observe the staining results by lightmicroscope directly, and quantitatively analyze staining by optical density.
2) Adjecent series of APP/PS1 brain tissure was stained myelin with gold chloride and axon with anti-neurofilament M145 antibody, we took corpus callosum and anteriorcommissure as representative regions of white matter. We measured the ROD(relativeoptical density) of representative regions, and quantitatively analyse myelinationdensity and axon density.
3) Evaluation of the extracellular amyloid load was only performed on the 24-months-oldAPP/PS1 mice as almost no Congo red positive deposits were observed in youngdouble transgenic mice.
Plaques loads were quantified using computer-based thresholding methods. Scanswere prepared using Photoshop CS2 to outline selected regions of interest. Imageswere then processed with ImageJ freeware(Rasband, W. S., Image J, U. S. NationalInstitutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2006)using a dedicated macrocommand that extracts amyloid deposits from backgroundtissue. Regional amyloid loads were expressed as percent of tissue surface stained bythe Congo red dye that corresponds to the proportion of plaques volume according toDelesse principle(Delesse, 1847). Amyloid loads were evaluated 1) in the whole brain, 2) in the rostral isocortex that is richly innervated by axons passing through theanterior corpus callosum, and 3) at the level of the two fibre tracts that wereinvestigated in the present study(anterior commissure, corpus callosum). Amyloidloads in the different regions were analyzed correlatively with the axon density andmyelin density in the corresponding regions and the atrophy of corpus callosum andanterior commissure.
4) Intraneuronal Aβimmunoreactivity was only quantified in the young APPxPS1 micebecause intracellular Aβstaining is absent in the aged mice of this line. Twosections/animals were selected at the level of the rostral cortex and a semi-quantitativeanalysis, based on a four points scale, was performed to evaluate levels of neuronalimmunostainings: 0: no obvious positive staining; 1: weak intracellular staining; 2:moderate staining; 3: strong staining.
5) Staining of degenerating neurons was performed using the Fluoro-Jade B dye with aslightly modified protocol. Glass-mounted sections were passed through absoluteethanol and 75% ethanol followed by a 1 minute rinse in distilled water. Tissue wasthen incubated in 0.06% potassium permanganate solution for 15 minutes with slight agitation and rinsed before staining in Fluoro-Jade B(Histo-Chem., Jefferson, AR;0.001% solution prepared in 0.1% acetic acid; 30 minutes at RT). After extensiverinsing in distilled water, sections were dehydrated, cleared in xylen and coverslipped.
Results
Myelin staining with improved gold chloride staining
7 days of rats are completely deficient of myelin staining. Quantitative analysis ofmyelination confirmed that myelination of 21 days old rats was higher than that of 14 daysold rats.
Pathological myelin staining was observed in APP/PS1 group. Myelination of APP/PS1mice was obviously lower than that of PS1 mol/Lice. There is conspicuous demyelinationin APP/PS1 group.
Altered volumes of fibre tracts in APPxPS1 mice
Gold chloride myelin staining, as compared to standard stains(e.g, HE or Nissl stains), wasallowed to precisely outline the area of the corpus callosum. In particular, delineating thecorpus callosum from adjacent white matter tracts(e.g. cingulate bundle, dorsal fornix, anddorsal hippocampal commissure) was greatly facilitated on myelin-stained sections. Laterallimits of the corpus callosum and borders of external capsula were identified by ahorizontal to vertical shift in fibre orientation. Also, the anterior commissure was easilyidentified and outlined from gold chloride-stained sections.
The size of the anterior commissure was similar in 2-months old PS1 mice andage-matched APPxPS1 transgenics(t(13)=-0.58, P＞0.05). On the contrary, the callosal sizewas significantly decreased in young APPxPS1 as compared to controls(t(13)=3.501,p＜0.005). Subregional analysis indicated a significant reduction in the size of the rostralcorpus callosum of young APPxPS1 mice (t(13)=3.743, p＜0.005) while there was nodifferences between genotypes in the surface area of the posterior corpus callosum(t(13)=0.136, P＞0.05).
With aging, a significant increase in white matter volumes was observed in PS1 control mice(corpus callosum: t(12)=3.858, p＜0.005; anterior commissure: t(12)=4.275,p＜0.005). This phenomenon was clearly not evidenced in the double APPxPS1 transgenics:in this genotype, the size of the corpus callosum remained constant between 2 and 24months(t(12)=1.850, P＞0.05) and surface area of the anterior commissure evenundergoes atrophy with aging(t(12)=2.284, p＜0.05). As a consequence, strong differencesbetween genotypes were observed in 24-months old mice with APPxPS1 transgenicsshowing, in comparison to PS1 controls, decreased white matters surface areas. This wasobserved at the level of the anterior commissure (t(11)=6.388, p＜0.0001) and of thecorpus callosum(total: t(11)=4.653, p＜.001; anterior: t(11)=5.404, p＜0.0005). The onlyposterior part of the corpus callosum did not show significant atrophy in old APPxPS1 mice(t(11)=1.492, P＞0.05).
Potentiation of axonal loss in old APPxPS1 mice
Axonal densities in the corpus callosum and anterior commissure were quantified usingROD analysis of neurofilament immunostainings.
In 2-months old mice axonal densities were similar in both genotypes whatever the fibretract considered(all p＞0.35). With aging, a severe decrease in neurofilament staining wasobserved, both in APPxPS1 and PS1 mice, testifying for axonal loss in the corpus callosumand anterior commissure(all p＜0.0001). Age-related reduction of neurofilament stainingwas however largely more pronounced in old APPxPS1 mice than in aged PS1 controls.Decreased axonal densities in old APPxPS1 mice was further confirmed in the differentsub-regions of the corpus callosum(all p＜0.0001) and also at the level of the anteriorcommissure(t(10)=4.16; p＜0.005).
Abnormal myelination in old APPxPS1 mice
ROD analysis of gold chloride stainings in 2-months old mice indicated comparable myelindensities in PS1 and APPxPS1 transgenics(corpus callosum: t(13)=0.318, P＞0.05;anterior commissure: t(13)=1.277, P＞0.05).
The myelination of the anterior commissure was not affected by aging(PS1: t(12)=1.292,P＞0.05; APPxPS1: t(12)=0.556, P＞0.05) and myelin densities in this fibre tract were similar in 24-months old PS1 and APPxPS1 mice (t(11)=0.679, P＞0.05). On the otherside, an increase of the myelination of the corpus callosum was observed when comparing2-months and 24-months old PS1 mice (t(12)=2.823, p＜0.05). Noticeably, myelinationbuild up with progressive aging was observed in control animals in the rostral corpuscallosum(t(12)=4.171, p＜0.005) but not in its posterior part(t(12)=0.461, P＞0.05).Contrarily to PS1 mice, such age-dependent callosal myelination was not observed inAPPxPS1 mice (t(12)=0.7, P＞0.05) and consequently decreased myelin staining wasevidenced in 24-months old APPxPS1 mice when compared to PS1 age-matched controls(total corpus callosum: t(11)=3.332, p＜0.01). Differences between genotypes were furtherconfirmed at the level of the anterior corpus callosum: (t(11)=3.512, p＜0.005) while nodifference between PS1 and APPxPS1 mice was evidenced in more caudal regions of thecorpus callosum (t(11)=1.9; P＞0.05).
Qualitative examination of myelin stained sections was then performed in old APPxPS1mice. No evidence of myelin breakdown(debris) was found in the large myelinated bundlesof the corpus callosum. However, in comparison to control animals, myelin appeared to befragmented in the isocortex and the hippocampus of APPxPS1 mice. Myelin material wasoften detected under the form of small tortuous segments with bead like varicosities. Thesemorphological anomalies, absent in young APPxPS1 mice, were found at the vicinity of Aβaggregation sites but also in the parenchyma in areas distant from plaques.
Relationship with Aβpathology and neurodegeneration
Congo red positive aggregates were detected and quantified in the anterior commissure(mean load=2.8%; min=1.6%; max=6%) and in the corpus callosum(mean load=2.2%;min=1.6%; max=2.7%) of old APPxPS1 mice. Correlative analysis did not revealsignificant associations between local amyloid loads in fibre tracts and white matteranomalies(decreased axonal densities and myelination: all p＞0.111). Also there were nocorrelations between morphology of the corpus callosum/anterior commissure and totalbrain or cortical amyloid loads (all p＞0.196).
In addition to amyloid plaques loads, intraneuronal Aβwas semi-quantitatively assessed inthe frontal cortex of young APPxPS1 mice. As expected from previous observations, positive labeling was detected using the 4G8 antibody in a subset of cells. Staining wasmainly observedin deep cortical layers(Ⅴ) involving a distinctive band of large pyramidalcells. However, there were no associations between levels of intracellular Aβthat maysignificantly vary from one animal to the other(mean=7.8; min=4.5; max=11.5) andaxonal and myelin markers(all p＞0.119).
The Fluoro-Jade B dye was used to assess neurodegeneration in APPxPS1 mice but nopositive neurons were detected in the studied animals(data not showed). In particular nodegenerating neurons were observed in the cortical layers with high densities of Aβpositiveneurons(see above). Only the core of amyloid deposits and surrounding degeneratingdystrophic neurites as well as reactive astrocytes were detected with Fluoro-Jade B in oldAPPxPS1 mice.
Conclusions
1) Improved myelin staining is rapid、simple、sensitive and stable, can be used for bothqualitative and quantitative analysis of myelination.
2) Gold chloride myelin staining allowed outlining precisely the shape of the corpuscallosum and measuring the size, which give a simple and delicate method to analyzequantitatively the atrophy of the corpus callosum.
3) There was conspicuous atrophy in the fiber tracts in the anterior brain of old APP/PS1mice, which is related with both axon loss and demyelination. However, thepotentiation of axon loss may be the first reason for the atrophy of fiber tracts.
4) The amyloid loads of the whole brain and the regions of interest didn't correlate withthe anormalies of fiber tracts, including axon loss and demyelination. That mean,extracellular amyloid beta doesn't have a clear pathogenicity.
5) Intracellular amyloid beta deposits are mostly located in the cortical layer V. the cells inCC, also the origins of callosal afferents, have been traced back to cell bodies in layerV. One may hypothesize that, early during aging, APPxPS1 mice accumulate Aβin asubset of cortical neurons that later become dysfunctional, develop axonal pathology(loss of neurofilament immunoreactivity and of myelin) and eventually die.
阿尔茨海默病(AD)是一种高发病率的神经系统变性疾病，临床上以逐渐进展且不可逆转的行为和认知障碍为特点。在AD进展过程中脑内病理学改变主要涉及2个方面：细胞外淀粉样蛋白β(Aβ)沉积形成的老年斑和细胞内tau蛋白积聚形成的神经纤维缠绕并最终促进细胞骨架的破坏、分解。目前AD的研究大部分是以一个重要的生理病理学假设所引导的，即所谓的“淀粉样蛋白瀑布学说”，该假说认为Aβ在脑内的沉积在AD疾病发生发展过程中扮演了一个最主要的角色，Aβ的沉积决定了脑内其他病理学改变的发生(如：突触和神经元的死亡)，并且最终导致了痴呆的临床阶段。其中最支持淀粉样蛋白瀑布学说的依据来源于对早发性家族性AD疾病的研究，早发性家族性AD与不同的基因突变有关：淀粉样蛋白前体蛋白基因(APP)和早老素1&2基因(PS1和PS2)，他们均与Aβ的生物合成有关。这些基因的机能障碍被认为导致APP代谢的异常从而最终导致Aβ过多产生。在表达突变的一个或几个上述基因的转基因小鼠中，我们观察到Aβ产生的增加和脑实质中淀粉样斑块的沉积，这些小鼠随后还出现了神经病理学的改变以及模拟AD表型的行为学的异常改变。
到目前为止，无论是在AD病人还是AD疾病动物模型中，脑内Aβ的沉积对临床痴呆症状的确切影响还没有找到直接的证据。临床病理学相关分析已经得到了一些有希望的结论：形成斑块之前的可溶性Aβ集合现在被认为是最具有毒性的，相反，积聚的不溶性Aβ集合致病性较弱。除此之外，Aβ的沉积和脑功能障碍的关系可能是间接的，他们被中间的神经病理学改变所调控。例如，AD病人脑内存在白质的异常，推测与脑-血管的异常相关，在模拟脑内淀粉样改变的转基因小鼠脑内，我们不仅能通过传统的神经病理学检测，而且能在体通过高新技术，如扩散增强成像技术，观察到该转基因小鼠脑内也存在白质的损害。最近有学者提出：白质缺陷可能是AD很重要的诊断性生物标记物。白质改变所带来的后果是显而易见的：神经网络的断开，随之而来纤维的丢失，最终导致皮层机能的紊乱；中枢神经系统髓鞘的崩解通过改变动作电位传播以及增加脑能量的输出，对神经元活动有致命的影响。
白质异常可能反映了轴突的丢失，也可能是脱髓鞘，并且确切地区分这两种过程十分困难。已有报道表明，AD病人不仅有轴突密度的下降，同时也存在髓鞘的崩解。
从AD动物模型的实验研究，可以明确地推出脑内Aβ的沉积可导致轴突的病理改变，这一点为联系脑内术期淀粉样改变和行为／认知障碍之间架起了一座桥梁。在本研究中，我们利用APP／PS1双转基因小鼠进一步评价了AD转基因小鼠白质的完整性。胼胝体和前联合在AD病人中有很大的改变，因此我们选择这两个区域作为白质的代表区域，通过神经纤维丝免疫组织化学染色分析了该区的轴突密度，通过氯化金染色评价了髓鞘的完整性；并且我们观察了APP／PS1小鼠白质的年龄相关的异常改变特点，进一步评价了这些异常改变和脑内淀粉样改变之间的关系。
方法
[1]对Schmued氯化金髓鞘染色进行了一些改动，并且采用该方法对不同年龄组大鼠的髓鞘染色进行定量分析，以及通过对不同表型小鼠的髓鞘染色进行定性分析。
[2]相邻的APP／PS1小鼠脑组织片分别采用氯化金髓鞘染色和抗神经微丝轴突染色，取胼胝体和前联合作为白质染色的代表区域，分别测量了代表区域平均吸光度值ROD，定量分析比较了该区域不同表型以及不同年龄小鼠之间髓鞘密度和轴突密度改变情况。
[3]细胞外淀粉样蛋白沉积采用标准的刚果红染色观察。采用基于计算机阈值方法，利用软件自动挑选有斑块的地方，根据Delesse原则计算刚果红染色区域占脑组织总面积的百分比，从而计算出淀粉样蛋白的含量。全脑、皮层运动区以及两个白质代表区域的淀粉样蛋白含量分别通过上述方法计算得出；然后分别对全脑和两个白质代表区域的淀粉样蛋白含量与该区域髓鞘密度、轴突密度以及代表区域面积大小进行了相关性分析。
[4]细胞内淀粉样蛋白通过4G8抗体进行染色，对阳性染色进行了半定量分析，其结果与前脑胼胝体面积大小进行相关性分析。
[5]神经元退行性变的评价采用Fluoro-Jade B染料染色方法，其结果与髓鞘以及轴突密度改变进行相关性分析。
结果
[1]新生7d大鼠髓鞘化染色阴性，21d大鼠的髓鞘化程度比14d大鼠的髓鞘化程度明显增高。APP／PS1组小鼠可观察到病理性髓鞘染色，定量分析显示其目标区域相对光密度值(ROD)低于PS1组，APP／PS1组存在明显的脱髓鞘改变。
[2]在脑组织冠状位切片上，氯化金髓鞘染色可以比较精确地确定胼胝体的轮廓，特别是将胼胝体与其周围相邻的白质纤维分开(例如：扣带回、背侧穹窿、背侧海马联合纤维)变得十分便捷；同时，胼胝体侧边的边缘和外囊的界限通过纤维不同方向走形达到区别。前联合在脑组织冠状位切片上很容易识别。
[3]APP／PS1小鼠纤维束体积的异常改变
在2月龄小鼠组，APP／PS1小鼠的前联合大小和PS1组小鼠的前联合大小是相当的(t(13)=-0.58，p＞0.05)；相反，APP／PS1组小鼠胼胝体大小比PS1组显著地减少了(t(13)=3.501，p＜0.005)。胼胝体不同亚区分析显示，胼胝体的减小主要集中在嘴部(t(13)=3.743，p＜0.005)。随着年龄的增长，PS1对照组白质体积有显著地增加(胼胝体：t(12)=3.858，p＜0.005；前联合：t(12)=4.275，p＜0.005)。然而，在APP／PS1转基因小鼠这种现象并不存在。胼胝体大小在2月龄和24月龄之间是相当的(t(12)=1.850，p＞0.05)，并且前联合的表面积随着年龄的增长不断地萎缩(t(12)=2.284，p＜0.05)。老年组APP／PS1小鼠前联合(t(11)=6.388，p＜0.0001)和胼胝体(胼胝体总：t(11)=4.653，p＜0.001；胼胝体嘴部：t(11)=5.404，p＜0.0005)比老年组PS1组小鼠前联合和胼胝体有显著地缩小。但是，在老年组胼胝体后部，PS1组和APP／PS1组之间没有区别(t(11)=1.492，p＞0.05)。
[4]老年组APP／PS1小鼠轴突丢失突出：在2月龄小鼠，2组小鼠的轴突密度都是相当的(p＞0.35)。随着年龄的增长，无论是在APP／PS1组还是PS1组，神经微丝染色都有严重的下降(p＜0.0001)，从胼胝体和前联合的统计学数据可以确定。然而，年龄相关的神经纤维染色的下降在老年组APP／PS1比老年组PS1组更加突出(p＜0.0001)。
[5]老年组APP／PS1小鼠异常的髓鞘染色定量分析：在2月龄组小鼠，氯化金染色的ROD分析显示PS1组和APP／PS1组的髓鞘密度是相当的(胼胝体：t(13)=0.318，p＞0.05；前联合：t(13)=1.277，p＞0.05)。
前联合的髓鞘化程度并没有随着年龄的影响而有所下降(PS1：t(12)=1.292，p＞0.05；APPxPS1：t(12)=0.556，p＞0.05)，因此在24月龄PS1组和APP／PS1组的前联合处髓鞘化密度是相当的(t(11)=0.679，p＞0.05)。然而，24月龄PS1小鼠胼胝体的髓鞘化程度比2月龄PS1有增高(t(12)=2.823，p＜0.05)，并且值得注意的是，随着年龄的增长，髓鞘化的程度主要发生在胼胝体的嘴部(t(12)=4.171，p＜0.005)，而在胼胝体后部并不存在这种情况(t(12)=0.461，p＞0.05)。和PS1小鼠相反，年龄相关的胼胝体不断髓鞘化过程并不存在于APP／PS1小鼠组(t(12)=0.7，p＞0.05)，因此24月龄APP／PS1小鼠的髓鞘化染色比24月龄PS1小鼠有明显的下降(总胼胝体：t(11)=3.332，p＜0.01)；并且这种髓鞘染色的下降主要位于胼胝体的嘴部(t(11)=3.512，p＜0.005)。在胼胝体后部PS1组和APP／PS1组之间没有区别(t(11)=1.9，p＞0.05)。
[6]光学显微镜下，老年组APP／PS1小鼠异常的髓鞘染色定性分析：髓鞘染色的定性分析检测，在老年组APP／PS1小鼠，在大的髓鞘化纤维束，没有明显的髓鞘崩解；但是和PS1对照组小鼠比较，APP／PS1小鼠的新皮层和海马区可以看到断裂的髓鞘碎片，髓鞘物质以小的扭曲的片段存在，并且带有“静脉曲张样改变”。这些形态学的异常(在年轻组APP／PS1小鼠组看不到)不仅出现在Aβ聚集的边缘，在远离Aβ斑块的脑组织实质中也可以看到。
[7]全脑及皮层兴趣区Aβ的含量和纤维束异常之间的关联分析在老年组APP／PS1组小鼠，定量测量了刚果红阳性的集聚物(前联合：Aβ平均含量=2.8％，最小值=1.6％，最大值=6％；胼胝体：Aβ平均含量=2.2％，最小值=1.6％，最大值=2.7％)。相关性分析表明，纤维束局部淀粉样蛋白含量和白质异常(包括轴突密度的下降和脱髓鞘)并不存在关联(所有的p＞0.111)。而且，全脑或皮层的淀粉样蛋白含量与胼胝体和前联合的大小之间也都没有关联(所有的p＞0.196)
[8]脑内皮质V层细胞内Aβ的含量的半定量测定和纤维束异常之间的关联分析在年轻组APP／PS1小鼠的前皮层，细胞内Aβ被半定量分析。和以往的观察一致，通过4G8抗体可以观察到阳性的染色。细胞内Aβ主要存在于皮层V。尽管细胞内Aβ水平在每个小鼠中半定量变化很大(平均值=7.8；最小值=4.5；最大值11.5)，但是相关性分析仍表明细胞内Aβ含量与轴突和髓鞘密度改变之间没有关联(所有p＞0.1119)。
[9]APP／PS1小鼠皮层变性神经元
采用Fluoro-Jade B染料观察APP／PS1小鼠神经元退行性变化。我们在研究动物中并未发现阳性的神经元染色。即使是在含有大量细胞内Aβ阳性皮层神经元中，我们也未发现退行性变化的神经元。仅仅在老年组APP／PS1小鼠，我们看能观察到淀粉样沉积的核心以及围绕在其周围退行性变化的神经突和反应性星形胶质细胞。
结论：
[1]改良的氯化金髓鞘染色快速、简单、敏感、稳定，可进行髓鞘化定性、定量分析。
[2]利用氯化金髓鞘染色，可以较精确地勾画胼胝体的轮廓并且测量其大小。为胼胝体大小的定量研究提供了一个简单而巧妙的方法。
[3]老年组APPxPS1前脑纤维束存在着严重萎缩，其发生不仅和轴突丢失有关，也和脱髓鞘有关。但APP／PS1小鼠严重的轴突丢失更加能解释纤维束的萎缩。
[4]无论是整个脑内还是局部脑细胞外淀粉样蛋白Aβ的含量，与老年组APP／PS1前脑纤维束的异常病理学改变，包括轴突的丢失和脱髓鞘导致的纤维束的萎缩，他们之间都不存在关联。即细胞外Aβ淀粉样蛋白并没有明确的致病性。
[5]细胞内β淀粉样蛋白的沉积，主要沉积皮层V，而该皮层细胞正好是胼胝体所对应的输入输出纤维的细胞体所在地，早期细胞内β淀粉样蛋白的沉积，随着年龄的增长，细胞会逐渐失去其功能，最终出现轴突丢失和脱髓鞘的病理学改变。
Alzheimer's disease(AD) is a high prevalence neurodegenerative disease accompanied bygradual and irreversible behavioral and cognitive impairments. Brain lesions observedduring the course of AD involve two main aspects: extracellular amyloid-beta(Aβ)deposition as senile plaques and intracellular tau accumulation forming neurofibrillarytangles and promoting cytoskeletal disorganization. Current research on AD is largelyguided by a dominant physiopathogenic hypothesis, the so-called amyloid cascade theory.Regularly commented on and amended, this model posits accumulation of Aβin thebrain, as a key primary event that determines the onset of other brain alterations(e.g.synaptic and neuronal death), finally leading to the clinical stage of dementia. Supportingthe amyloid cascade hypothesis, early-onset familial forms of AD are associated withmutations in different genes(Amyloid Precursor Protein(APP) and Presenilins 1&2,(PS1&2)) involved in the biosynthesis of Aβ. Dysfunction of these genes is logicallythought to compromise the normal catabolism of APP resulting in exaggerated Aβproduction. Increased Aβproduction and parenchymal amyloid plaques are indeeddescribed in transgenic mice overexpressing one or more of these mutated genes. Thesemice subsequently develop neuropathological alterations and behavioural impairmentsmimicking AD phenotype.
The exact impact of brain Aβaccumulation on clinical symptoms remains to-date difficultto decipher, both in AD patients and in animal models of the disease. Clinico-pathologicalcorrelative analyses have led to mitigated conclusions and it is now considered thatpre-plaques Aβassemblies are the most deleterious species while aggregated insolubledeposits have a reduced pathogenicity. In addition, the relationship between Aβaccumulation and brain dysfunction might be indirect and mediated by secondaryneuropathological alterations. For instance, white matter anomalies are described in ADpatients, presumably in association with cerebro-vascular impairments, and in transgenicmice modeling brain amyloidosis. They can be detected through conventionalpostmortem neuropathological examination, but also in vivo by means of dedicatedtechniques such as diffusion tensor imaging and it has recently been proposed that white matter defects are potent diagnostic biomarkers for AD. The functionalconsequences of altered white matter are obvious: Disconnection of neural networks occurfollowing fibres loss, leading to diaschesis and cortical disorganized activity. Also,disruption of myelin in the CNS white matter might have deleterious effects on neuronalcommunication by altering propagation of action potentials and increasing brain energyexpenditure.
Importantly, white matter anomalies might reflect either loss of fibers and/or demyelinationbut deciphering between the two processes could sometimes be hazardous. In particular, ithas been demonstrated that AD patients show concurrent decreased axonal densities andmyelin breakdown.
It is clear, especially from experimental studies in AD animal models, that axonal pathologycan be driven by Aβdeposition in the brain therefore possibly bridging the gapbetween amyloidosis and behavioural/cognitive impairments.
The aim of the present study was to further evaluate white matter integrity in a doubleAPPxPS1 mouse transgenic model with aggressive Aβ-related pathology. Twoaxonal bundles(corpus callosum and anterior commissure) that show significant alterationsin AD patients were selected and analyzed: axonal densities were quantified by meansof anti-neurofilament immunostainings and myelin integrity was evaluated byhistochemistry using a gold chloride staining that provides high contrast and spatialresolution. Age-dependent anomalies were detected in the white matter of APPxPS1mice and we further assessed the relationship between these alterations and Aβdeposition.
Methods
1) Three groups of new-born rats, each group contained 5 rats, which were 7 days、14days and 21 days old. The other 13 Alzheimer's disease transgenic rats, contained 5APP/PS1 mol/Lice and PS1 mol/Lice. Following decapitation the brains were extractedand treated. The brains were cut by the microtome. The brain tissue was stained byimproved gold chloride myelin staining. Observe the staining results by lightmicroscope directly, and quantitatively analyze staining by optical density.
2) Adjecent series of APP/PS1 brain tissure was stained myelin with gold chloride and axon with anti-neurofilament M145 antibody, we took corpus callosum and anteriorcommissure as representative regions of white matter. We measured the ROD(relativeoptical density) of representative regions, and quantitatively analyse myelinationdensity and axon density.
3) Evaluation of the extracellular amyloid load was only performed on the 24-months-oldAPP/PS1 mice as almost no Congo red positive deposits were observed in youngdouble transgenic mice.
Plaques loads were quantified using computer-based thresholding methods. Scanswere prepared using Photoshop CS2 to outline selected regions of interest. Imageswere then processed with ImageJ freeware(Rasband, W. S., Image J, U. S. NationalInstitutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2006)using a dedicated macrocommand that extracts amyloid deposits from backgroundtissue. Regional amyloid loads were expressed as percent of tissue surface stained bythe Congo red dye that corresponds to the proportion of plaques volume according toDelesse principle(Delesse, 1847). Amyloid loads were evaluated 1) in the whole brain, 2) in the rostral isocortex that is richly innervated by axons passing through theanterior corpus callosum, and 3) at the level of the two fibre tracts that wereinvestigated in the present study(anterior commissure, corpus callosum). Amyloidloads in the different regions were analyzed correlatively with the axon density andmyelin density in the corresponding regions and the atrophy of corpus callosum andanterior commissure.
4) Intraneuronal Aβimmunoreactivity was only quantified in the young APPxPS1 micebecause intracellular Aβstaining is absent in the aged mice of this line. Twosections/animals were selected at the level of the rostral cortex and a semi-quantitativeanalysis, based on a four points scale, was performed to evaluate levels of neuronalimmunostainings: 0: no obvious positive staining; 1: weak intracellular staining; 2:moderate staining; 3: strong staining.
5) Staining of degenerating neurons was performed using the Fluoro-Jade B dye with aslightly modified protocol. Glass-mounted sections were passed through absoluteethanol and 75% ethanol followed by a 1 minute rinse in distilled water. Tissue wasthen incubated in 0.06% potassium permanganate solution for 15 minutes with slight agitation and rinsed before staining in Fluoro-Jade B(Histo-Chem., Jefferson, AR;0.001% solution prepared in 0.1% acetic acid; 30 minutes at RT). After extensiverinsing in distilled water, sections were dehydrated, cleared in xylen and coverslipped.
Results
Myelin staining with improved gold chloride staining
7 days of rats are completely deficient of myelin staining. Quantitative analysis ofmyelination confirmed that myelination of 21 days old rats was higher than that of 14 daysold rats.
Pathological myelin staining was observed in APP/PS1 group. Myelination of APP/PS1mice was obviously lower than that of PS1 mol/Lice. There is conspicuous demyelinationin APP/PS1 group.
Altered volumes of fibre tracts in APPxPS1 mice
Gold chloride myelin staining, as compared to standard stains(e.g, HE or Nissl stains), wasallowed to precisely outline the area of the corpus callosum. In particular, delineating thecorpus callosum from adjacent white matter tracts(e.g. cingulate bundle, dorsal fornix, anddorsal hippocampal commissure) was greatly facilitated on myelin-stained sections. Laterallimits of the corpus callosum and borders of external capsula were identified by ahorizontal to vertical shift in fibre orientation. Also, the anterior commissure was easilyidentified and outlined from gold chloride-stained sections.
The size of the anterior commissure was similar in 2-months old PS1 mice andage-matched APPxPS1 transgenics(t(13)=-0.58, P＞0.05). On the contrary, the callosal sizewas significantly decreased in young APPxPS1 as compared to controls(t(13)=3.501,p＜0.005). Subregional analysis indicated a significant reduction in the size of the rostralcorpus callosum of young APPxPS1 mice (t(13)=3.743, p＜0.005) while there was nodifferences between genotypes in the surface area of the posterior corpus callosum(t(13)=0.136, P＞0.05).
With aging, a significant increase in white matter volumes was observed in PS1 control mice(corpus callosum: t(12)=3.858, p＜0.005; anterior commissure: t(12)=4.275,p＜0.005). This phenomenon was clearly not evidenced in the double APPxPS1 transgenics:in this genotype, the size of the corpus callosum remained constant between 2 and 24months(t(12)=1.850, P＞0.05) and surface area of the anterior commissure evenundergoes atrophy with aging(t(12)=2.284, p＜0.05). As a consequence, strong differencesbetween genotypes were observed in 24-months old mice with APPxPS1 transgenicsshowing, in comparison to PS1 controls, decreased white matters surface areas. This wasobserved at the level of the anterior commissure (t(11)=6.388, p＜0.0001) and of thecorpus callosum(total: t(11)=4.653, p＜.001; anterior: t(11)=5.404, p＜0.0005). The onlyposterior part of the corpus callosum did not show significant atrophy in old APPxPS1 mice(t(11)=1.492, P＞0.05).
Potentiation of axonal loss in old APPxPS1 mice
Axonal densities in the corpus callosum and anterior commissure were quantified usingROD analysis of neurofilament immunostainings.
In 2-months old mice axonal densities were similar in both genotypes whatever the fibretract considered(all p＞0.35). With aging, a severe decrease in neurofilament staining wasobserved, both in APPxPS1 and PS1 mice, testifying for axonal loss in the corpus callosumand anterior commissure(all p＜0.0001). Age-related reduction of neurofilament stainingwas however largely more pronounced in old APPxPS1 mice than in aged PS1 controls.Decreased axonal densities in old APPxPS1 mice was further confirmed in the differentsub-regions of the corpus callosum(all p＜0.0001) and also at the level of the anteriorcommissure(t(10)=4.16; p＜0.005).
Abnormal myelination in old APPxPS1 mice
ROD analysis of gold chloride stainings in 2-months old mice indicated comparable myelindensities in PS1 and APPxPS1 transgenics(corpus callosum: t(13)=0.318, P＞0.05;anterior commissure: t(13)=1.277, P＞0.05).
The myelination of the anterior commissure was not affected by aging(PS1: t(12)=1.292,P＞0.05; APPxPS1: t(12)=0.556, P＞0.05) and myelin densities in this fibre tract were similar in 24-months old PS1 and APPxPS1 mice (t(11)=0.679, P＞0.05). On the otherside, an increase of the myelination of the corpus callosum was observed when comparing2-months and 24-months old PS1 mice (t(12)=2.823, p＜0.05). Noticeably, myelinationbuild up with progressive aging was observed in control animals in the rostral corpuscallosum(t(12)=4.171, p＜0.005) but not in its posterior part(t(12)=0.461, P＞0.05).Contrarily to PS1 mice, such age-dependent callosal myelination was not observed inAPPxPS1 mice (t(12)=0.7, P＞0.05) and consequently decreased myelin staining wasevidenced in 24-months old APPxPS1 mice when compared to PS1 age-matched controls(total corpus callosum: t(11)=3.332, p＜0.01). Differences between genotypes were furtherconfirmed at the level of the anterior corpus callosum: (t(11)=3.512, p＜0.005) while nodifference between PS1 and APPxPS1 mice was evidenced in more caudal regions of thecorpus callosum (t(11)=1.9; P＞0.05).
Qualitative examination of myelin stained sections was then performed in old APPxPS1mice. No evidence of myelin breakdown(debris) was found in the large myelinated bundlesof the corpus callosum. However, in comparison to control animals, myelin appeared to befragmented in the isocortex and the hippocampus of APPxPS1 mice. Myelin material wasoften detected under the form of small tortuous segments with bead like varicosities. Thesemorphological anomalies, absent in young APPxPS1 mice, were found at the vicinity of Aβaggregation sites but also in the parenchyma in areas distant from plaques.
Relationship with Aβpathology and neurodegeneration
Congo red positive aggregates were detected and quantified in the anterior commissure(mean load=2.8%; min=1.6%; max=6%) and in the corpus callosum(mean load=2.2%;min=1.6%; max=2.7%) of old APPxPS1 mice. Correlative analysis did not revealsignificant associations between local amyloid loads in fibre tracts and white matteranomalies(decreased axonal densities and myelination: all p＞0.111). Also there were nocorrelations between morphology of the corpus callosum/anterior commissure and totalbrain or cortical amyloid loads (all p＞0.196).
In addition to amyloid plaques loads, intraneuronal Aβwas semi-quantitatively assessed inthe frontal cortex of young APPxPS1 mice. As expected from previous observations, positive labeling was detected using the 4G8 antibody in a subset of cells. Staining wasmainly observedin deep cortical layers(Ⅴ) involving a distinctive band of large pyramidalcells. However, there were no associations between levels of intracellular Aβthat maysignificantly vary from one animal to the other(mean=7.8; min=4.5; max=11.5) andaxonal and myelin markers(all p＞0.119).
The Fluoro-Jade B dye was used to assess neurodegeneration in APPxPS1 mice but nopositive neurons were detected in the studied animals(data not showed). In particular nodegenerating neurons were observed in the cortical layers with high densities of Aβpositiveneurons(see above). Only the core of amyloid deposits and surrounding degeneratingdystrophic neurites as well as reactive astrocytes were detected with Fluoro-Jade B in oldAPPxPS1 mice.
Conclusions
1) Improved myelin staining is rapid、simple、sensitive and stable, can be used for bothqualitative and quantitative analysis of myelination.
2) Gold chloride myelin staining allowed outlining precisely the shape of the corpuscallosum and measuring the size, which give a simple and delicate method to analyzequantitatively the atrophy of the corpus callosum.
3) There was conspicuous atrophy in the fiber tracts in the anterior brain of old APP/PS1mice, which is related with both axon loss and demyelination. However, thepotentiation of axon loss may be the first reason for the atrophy of fiber tracts.
4) The amyloid loads of the whole brain and the regions of interest didn't correlate withthe anormalies of fiber tracts, including axon loss and demyelination. That mean,extracellular amyloid beta doesn't have a clear pathogenicity.
5) Intracellular amyloid beta deposits are mostly located in the cortical layer V. the cells inCC, also the origins of callosal afferents, have been traced back to cell bodies in layerV. One may hypothesize that, early during aging, APPxPS1 mice accumulate Aβin asubset of cortical neurons that later become dysfunctional, develop axonal pathology(loss of neurofilament immunoreactivity and of myelin) and eventually die.
引文
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