Wirenfeldt Martin, Dalmau Ishar, Finsen Bente
Anatomy and Neurobiology, University of Southern Denmark, Odense C, Denmark.
Glia. 2003 Nov;44(2):129-39. doi: 10.1002/glia.10277.
Stereology offers a set of unbiased principles to obtain precise estimates of total cell numbers in a defined region. In terms of microglia, which in the traumatized and diseased CNS is an extremely dynamic cell population, the strength of stereology is that the resultant estimate is unaffected by shrinkage or expansion of the tissue. The optical fractionator technique is very efficient but requires relatively thick sections (e.g., > or =20 microm after coverslipping) and the unequivocal identification of labeled cells throughout the section thickness. We have adapted our protocol for Mac-1 immunohistochemical visualization of microglial cells in thick (70 microm) vibratome sections for stereological counting within the murine hippocampus, and we have compared the staining results with other selective microglial markers: the histochemical demonstration of nucleotide diphosphatase (NDPase) activity and the tomato lectin histochemistry. The protocol gives sections of high quality with a final mean section thickness of >20 microm (h=22.3 microm +/- 0.64 microm), and with excellent rendition of Mac-1+ microglia through the entire height of the section. The NDPase staining gives an excellent visualization of microglia, although with this thickness, the intensity of the staining is too high to distinguish single cells. Lectin histochemistry does not visualize microglia throughout the section and, accordingly, is not suited for the optical fractionator. The mean total number of Mac-1+ microglial cells in the unilateral dentate gyrus of the normal young adult male C57BL/6 mouse was estimated to be 12,300 (coefficient of variation (CV)=0.13) with a mean coefficient of error (CE) of 0.06. The perspective of estimating microglial cell numbers using stereology is to establish a solid basis for studying the dynamics of the microglial cell population in the developing and in the injured, diseased and normal adult CNS.
体视学提供了一套无偏倚的原则，用于精确估计特定区域内的细胞总数。对于小胶质细胞而言，在创伤性和患病的中枢神经系统中，它是一个极具动态变化的细胞群体，体视学的优势在于所得估计不受组织收缩或扩张的影响。光学分选技术非常高效，但需要相对较厚的切片（例如，封片后≥20微米），并且要在整个切片厚度上明确识别标记细胞。我们已调整方案，用于在厚（70微米）的振动切片机切片中对小鼠海马体内的小胶质细胞进行Mac-1免疫组织化学可视化，以便进行体视学计数，并且我们已将染色结果与其他选择性小胶质细胞标记物进行了比较：核苷酸二磷酸酶（NDPase）活性的组织化学显示和番茄凝集素组织化学。该方案可提供高质量的切片，最终平均切片厚度>20微米（h = 22.3微米±0.64微米），并且在整个切片高度上对Mac-1 +小胶质细胞有出色的呈现。NDPase染色能很好地显示小胶质细胞，尽管在此厚度下，染色强度过高以至于无法区分单个细胞。凝集素组织化学不能在整个切片中显示小胶质细胞，因此不适合用于光学分选。正常年轻成年雄性C57BL / 6小鼠单侧齿状回中Mac-1 +小胶质细胞的平均总数估计为12,300个（变异系数（CV）= 0.13），平均误差系数（CE）为0.06。使用体视学估计小胶质细胞数量的前景是为研究发育中的、受伤的、患病的和正常成年中枢神经系统中小胶质细胞群体的动态变化建立坚实的基础。
