运用新的体视学方法定量研究大鼠海马结构神经元和突触数目

Quantitative study of neurons and synapses in rat hippocampus by new stereological methods

目的建立定量研究海马结构神经元数目和突触数目的无偏体视学方法。方法选择18月龄正常雌性Long-Evans大鼠5只,取一侧大脑半球进行石蜡包埋后切取14μm厚的冠状连续切片。根据均匀系统随机抽样的原则,在包含海马结构的连续切片中均匀随机地抽取切片。将抽取的一组切片进行神经元的组织化学染色,以计数神经元的数目;将抽取的另外一组切片进行突触的免疫组织化学染色,以计数突触的数目。在体视学设备下,计数大鼠海马结构CA1和DG的神经元数目和大鼠海马结构CA1、CA1放射层(CA1sr)、齿状回内分子层(DGiml)和齿状回外分子层(DGoml)的突触数目。结果得到大鼠海马结构CA1和DG的神经元数目分别为[(3.17×105)±(2.23×104)]和[(1.70×106)±(1.87×105)]。大鼠海马结构CA1、CA1 CA1 sr、DGiml和DGoml的突触数目分别为[(1.04×108)±(5.39×106)]、[(4.36×107)±(3.78×106)]、[(1.87×107)±(1.81×106)]和[(4.48×107)±(7.29×105)]。结论 本研究建立了定量研究海马结构各区域内神经元数目和突触数目的方法,可为定量研究正常老年及各种神经系统退行性疾病状态下海马结构各区域内神经元数目、突触数目及神经元与突触数目关系的改变提供方法学依据。

Objective To establish stereological methods for the quantitative studies of neurons and synapses in the hippocampus.Methods After five 18-month female Long-Evans rats were anesthesized,one of the two hemispheres was sampled randomly after fixation with paraformaldehyde perfusion,and then embedded in paraffin blocks.Serial coronal sections in thickness of 14 μm were cut.The sections were uniformly and randomly sampled from the consecutive sections through hippocampus according to the principles of systematic,uniform and random sampling.One series of sections were stained for Nissl substance for the quantification of neuron number,and another series of sections were stained for the quantification of synaptophysin-positive bouton number.The number of neurons in CA1 and DG and the number of synaptophysin-positive boutons in CA1,CA1sr,DGiml,and DGoml were estimated with the unbiased stereological technique,the optical fractionator.Results The total number of the neurons in CA1 and DG was 3.17×10^5±2.23×104,and 1.70×10^6±1.87×10^5,respectively.The total number of the synapses in CA1,CA1sr,DGiml,and DGoml was 1.04×10^8±5.39×10^6,4.36×10^7±3.78×10^6,1.87×10^7±1.81×10^6,and 4.48×10^7±7.29×10^5,respectively.Conclusion Our established methods can quantitatively study the neuron and synapse changes during hippocampal formation in normal aging process and in various neurodegenerative diseases.