Estimation of absolute microglial cell numbers in mouse fascia dentata using unbiased and efficient stereological cell counting principles
Estimation of absolute microglial cell numbers in mouse fascia dentata using unbiased and efficient stereological cell counting principles
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作者：
Wirenfeldt, M;Dalmau, I;Finsen, B
立体学提供了一套无偏的原则，以获得精确估计总细胞数在一个确定的区域。就小胶质细胞而言，在创伤和病变的中枢神经系统中是一个极其动态的细胞群，体视学的强度是由此产生的估计不受组织收缩或扩张的影响。光学分馏仪技术非常有效，但需要相对较厚的切片（例如，覆盖后大于或等于20 μ m），并且在整个切片厚度上明确识别标记细胞。我们采用了我们的方案，对小鼠海马内厚（70 μ m）振动体切片的小胶质细胞进行Mac-1免疫组织化学可视化，用于体视学计数，并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）活性的组织化学证明和番茄凝集素组织化学。该方案给出了高质量的切片，最终平均切片厚度为bbb20 μ m (h = 22.3 μ m +/- 0.64 μ m)，并且在整个切片高度上都有Mac-1（+）小胶质细胞的良好表现。NDPase染色可以很好地显示小胶质细胞，尽管在这种厚度下，染色强度太高，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，不适合光学分馏仪。正常成年雄性C57BL/6小鼠单侧齿状回Mac-1（+）小胶质细胞的平均总数估计为12300个（变异系数(CV) = 0.13），平均误差系数（CE）为0.06。利用体视学方法估计小胶质细胞数量，为研究发育中、损伤、病变和正常成人中枢神经系统中小胶质细胞群的动态变化奠定了坚实的基础。(C) 2003 Wiley-Liss, Inc。
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., greater than or equal to20 mum 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 mum) 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 mum (h = 22.3 mum +/- 0.64 mum), 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. (C) 2003 Wiley-Liss, Inc.