背景:Cavalieri估计器用于大脑和大脑区域的体积测量。从这个估计器导出的是面积分数分割器(AFF),用于小二维元素的有效面积和数量估计,比如横切神经的轴突.然而,根据我们的知识,AFF尚未与连续切片分析相结合来测量小尺寸神经结构的体积。
方法:以黑质纹状体多巴胺能系统为例,我们描述了一个基于Cavalieri原理和AFF的协议来估计其躯体的体积,核,树突状,成年小鼠的轴突和轴突末端细胞区室。该方案包括(1)对感兴趣区域(黑质,黑质纹状体,尾状壳核),(2)现场的共焦图像采集,(3)使用Cavalieri的2D点计数网格标记细胞域,和4)使用每个隔间的估计面积确定隔间的总体积,和截面之间的距离。
结果:黑质纹状体系统的体积为〜0.38mm3,其中〜5%对应于周围核和细胞核,~10%到神经纤维/树突,〜85%的轴突和静脉曲张。
方法:与其他测量离散物体体积的方法相比,例如光学成核剂或3D重建,它以其多功能性和易用性而脱颖而出。
结论:使用简单的定量,评估系统整体状态的无偏倚方法可以量化可能伴随神经退行性过程的隔室特异性变化.
BACKGROUND: The Cavalieri estimator is used for volume measurement of brain and brain regions. Derived from this estimator is the Area Fraction Fractionator (AFF), used for efficient area and number estimations of small 2D elements, such as axons in cross-sectioned nerves. However, to our knowledge, the AFF has not been combined with serial sectioning analysis to measure the volume of small-size nervous structures.
METHODS: Using the nigrostriatal dopaminergic system as an illustrative case, we describe a protocol based on Cavalieri\'s principle and AFF to estimate the volume of its somatic, nuclear, dendritic, axonal and axon terminal cellular compartments in the adult mouse. The protocol consists of (1) systematic random sampling of sites within and across sections in regions of interest (substantia nigra, the nigrostriatal tract, caudate-putamen), (2) confocal image acquisition of sites, (3) marking of cellular domains using Cavalieri\'s 2D point-counting grids, and 4) determination of compartments\' total volume using the estimated area of each compartment, and between-sections distance.
RESULTS: The volume of the nigrostriatal system per hemisphere is ∼0.38 mm3, with ∼5 % corresponding to perikarya and cell nuclei, ∼10 % to neuropil/dendrites, and ∼85 % to axons and varicosities.
METHODS: In contrast to other methods to measure volume of discrete objects, such as the optical nucleator or 3D reconstructions, it stands out for its versatility and ease of use.
CONCLUSIONS: The use of a simple quantitative, unbiased approach to assess the global state of a system may allow quantification of compartment-specific changes that may accompany neurodegenerative processes.