Abstract
Optical fractionators have dominated the field of neural cell counting for two decades. These unbiased stereological techniques are often used for the quantification of hippocampal cell proliferation in neurogenesis experiments. However, the heterogeneous distribution of labeled cells, especially in the form of clusters, confounds the application of these techniques. A critical evaluation of the applicability of the optical fractionator suggests that absolute counting achieves higher efficiency in the quantification of cell proliferation than unbiased estimations.
The discovery that new neurons are continuously produced throughout adulthood (adult neurogenesis) in discrete regions of the brain, notably within the dentate gyrus of the hippocampus and the olfactory bulb, has led to an explosion of studies aimed at elucidating the biological significance of this phenomenon as well as the factors that might impact this process. Neurogenesis is a complex, multi-step process, consisting of neural progenitor cell proliferation, differentiation, migration, neuronal maturation, and cell death (Ehninger and Kempermann, 2008). The production of new neurons can be modulated at multiple levels. Since cell proliferation represents the first critical step toward neurogenesis, many studies have focused primarily on this process or, at the very least, have included an examination of cell proliferation as part of a larger, comprehensive investigation on neurogenesis. Cell proliferation in these studies is usually measured by injecting the thymidine analog 5-bromo-2′-deoxyuridine (BrdU), which becomes incorporated into newly synthesized DNA of cells during the S phase of the cell cycle. After an appropriate post-injection survival period (e.g., 2 h), animals are sacrificed and their brains are then processed for BrdU immunohistochemistry. Another common, reliable method for assessing cell proliferation is to stain for the endogenous nuclear protein Ki67, which is expressed during all phases of the active cell cycle (G1, S, G2, and mitosis), but is absent during the resting (G0) phase (Kee et al., 2002). In the dentate gyrus of the hippocampus, newly proliferating cells are found predominantly in the subgranular zone (SGZ), where the neural progenitors reside, and often appear in clusters (Wojtowicz and Kee, 2006).
The optical fractionator combines two stereological components: a three-dimensional probe for counting cell nuclei, the optical dissector, and a sampling scheme, the fractionator. The optical dissector provides unbiased estimates of cell numbers independent of assumptions on the size and shape of the cells. Its value converges to the true number of cells as the number of samples increases. The fractionator involves sampling a known fraction of a structural component or region (e.g., the SGZ of the dentate gyrus). The number of cells (N) is then estimated as:
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where yes is the total number of cells actually counted in the dissectors; asf, the area sampling fraction, is the area of the counting frame relative to the area associated with each x, y movement; and ssf, the section sampling fraction, is the fraction of total sections sampled; and finally, the thickness sampling fraction tsf captures the part of the investigated cross-sectional area of the sampled sections (Gundersen et al., 1988; West et al., 1988, 1991, West and Gundersen, 1990). The optical fractionator has dominated the field of unbiased estimations of cell number and has been extensively used to quantify the number of cells in different brain structures. However, the application of this technique in neurogenesis studies is not rigorous.
Although this estimator is independent of assumptions on the size and shape of the cells, it is not free of assumptions about the structure of the tissue. Indeed, the efficiency of this technique depends on the homogeneity of the cell distribution in the region of interest. The distribution of proliferating cells in the SGZ of the hippocampus is not regular (Figure 1A). Depending on the size of the area of the counting frame (acf) of the dissector, there is a probability that the dissector might miss the proliferating cells. Furthermore, proliferating cells in the SGZ commonly appear in clusters and these clusters convert the approximately homogenous distribution of labeled cells in the hippocampus to a non-uniform and heterogeneous pattern (Figure 1B).
