Therefore, area K2 represents the Ki-67 and PI double positive nuclei of newly formed cells
Therefore, area K2 represents the Ki-67 and PI double positive nuclei of newly formed cells. two stable regions of mitotic activity, the subventricular zone (SVZ) of the lateral ventricle in the frontal cortex and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus (1,2). Active neurogenesis in hippocampi lead to the incorporation of thousands of new granule cells into the dentate gyrus every day (3). While the regenerative potential of the mammalian brain is sustained throughout the life span, the magnitude of the proliferative efficacy of neural progenitors declines with age and diseases, such as Alzheimer’s disease (AD) (4-7). Therefore, to reverse and/or to prevent from neurogenic deficits becomes a potential therapeutic strategy for anti-neurodegenerative diseases, including AD. For example, extensive efforts have been made to experimentally evaluate the efficiency of potential neurogenic enhancers using transgenic mouse AD models (8-11). The most common method forin vivoanalysis of neurogenesis is the unbiased stereology of 5-bromo-2-deoxyuridine (BrdU) immunohistochemical labeled serial brain sections under microscopy, which are both labor and time extensive. In addition, stereological analysis uses the optical fractionator (12), a combination of optical dissector with statistically optimized spatial sampling protocols, where the estimates are obtained from cell densities, must be restricted to well defined structures of isotropic architecture and measurable volume (12). Moreover, the actual positive cell numbers are achieved by multiplying cell density by volume, which is determined by precisely drawing the structural boundary and accurately estimating the tissue volume change during section preparation. The numbers obtained are not independent variables and therefore are limited statistically to compare against volume (13). Thus, extreme importance is placed on establishing a high throughput evaluation forin vivoneurogenic efficiency screening that can be completed in a short time and used for a large sample size. Of more objective importance in particular, is the development of potential neurogenic drugs. The thymidine analog, BrdU, is a commonly used molecule to measure cell proliferation in different tissues, including the CNS, based on the stable incorporation occurring in S-phase of the cell cycle (3). However, BrdU is toxic to newborn neurons and triggers cell death by altering DNA stability and lengthening the cell cycle. Additionally DW-1350 BrdU has various mitogenic, transcriptional, and translational effects on cells that incorporate the nucleoside (14). Therefore, difficulty is found in giving a clear interpretation of the 5 times less amount of BrdU positive cells in mice 21 days after BrdU injection than that detected 24 hours after BrdU injection (11,15). In determining whether the apoptosis of the newly formed DW-1350 cells is a natural phenomenon or is triggered by the BrdU incorporation, a recentin vitrostudy, in which human neural progenitor cells were used, reported that BrdU doses in the concentration range that is recommended for cell proliferation studies (1-10 M) interfered with the survival of newborn neurons (BrdU/TuJ1+ cells), and high doses of BrdU activated the classical DW-1350 apoptosis pathways in newly formed neurons (16). When administered to FAAP24 pregnant mice and rats, BrdU interfered with embryonic brain development, caused bodily defects in embryos, and caused postnatal behavioral abnormalities (17). In addition, BrdU is not only a marker of the S-phase of the cell cycle but is also a marker of DNA synthesis, including DNA repair, and that, on the other hand, may induce a false positive. Therefore, importance is placed on using a less toxic and efficient molecule, ideally an endogenous marker, to probe neurogenesis in establishing a high throughput screen. In this study, we reported a high throughput flow cytometric assay to evaluate the newly formed cells in rodent hippocampi within different conditions. The assay analyzed immunolabeled fluorescent Ki-67, an endogenous protein only expressed in active cell cycles (18-21), positive cells in homogeneous, isotropic suspensions. Hippocampi were first dissected from fixed brain hemispheres. The isotropic nuclear suspensions were then extracted, and immuno-labeling of the newly formed cells by cell proliferation marker Ki-67 was completed. Finally the positive fluorescent cells were analyzed by flow cytometry. == 2. Materials and Methods == == 2.1. Animal == Ovariectomy reduction DW-1350 of hippocampal neurogenesis was demonstrated, and estradiol reversed this decrease (22,23). Therefore, we used female ovariectomized (OVX) mice to evaluate our methods. Female C57/B6 mice were purchased from Harlan Laboratories, Indianapolis, IN). Animals were ovariectomized and the estradiol.