The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis

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Abstract

Adult animals continue to produce new neurons in the dentate gyrus of hippocampus. Until now, the principal method of studying neurogenesis has been to inject either tritiated thymidine or 5'-Bromo-2-deoxyuridine (BrdU) intraperitoneally followed by autoradiographic or immunohistochemical detection methods respectively. However, such exogenous markers may produce toxic effects. Our objective was to determine whether Ki-67, a nuclear protein expressed in all phases of the cell cycle except the resting phase, can be used as an alternative, endogenous marker. Using immunohistochemistry, we examined Ki-67 and BrdU expression pattern in rats. Ki-67 was expressed within the proliferative zone of the dentate gyrus and its expression pattern mimicked that of BrdU when examined soon after exogenous BrdU administration. Quantitative comparison of BrdU and Ki-67-positive cells showed 50% higher numbers of the latter when examined 24 h after the BrdU injection. This was expected, since BrdU can be incorporated into DNA only during the S-phase of the mitotic process, whereas Ki-67 is expressed for its whole duration. Experimental increases (by ischemia) or reductions (by radiation) in the number of mitotic cells produced parallel changes in BrdU and Ki-67 signals. Thus, Ki-67 is an effective mitotic marker and has most of the benefits of BrdU and none of the costs. This study provides evidence for Ki-67 to be used as a marker of proliferation in the initial phase of adult neurogenesis.

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Vascular niche for adult hippocampal neurogenesis.

Theo Palmer, Andrew R. Willhoite, Fred Gage (2000)

The thin lamina between the hippocampal hilus and granule cell layer, or subgranule zone (SGZ), is an area of active proliferation within the adult hippocampus known to generate new neurons throughout adult life. Although the neuronal fate of many dividing cells is well documented, little information is available about the phenotypes of cells in S-phase or how the dividing cells might interact with neighboring cells in the process of neurogenesis. Here, we make the unexpected observation that dividing cells are found in dense clusters associated with the vasculature and roughly 37% of all dividing cells are immunoreactive for endothelial markers. Most of the newborn endothelial cells disappear over several weeks, suggesting that neurogenesis is intimately associated with a process of active vascular recruitment and subsequent remodeling. The present data provide the first evidence that adult neurogenesis occurs within an angiogenic niche. This environment may provide a novel interface where mesenchyme-derived cells and circulating factors influence plasticity in the adult central nervous system. Copyright 2000 Wiley-Liss, Inc.

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Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication.

H G Gratzner (1982)

Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

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Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat.

H.A. Cameron, C.S. Woolley, B.S. McEwen … (1993)

In order to determine whether newly born cells in the dentate gyrus of the adult rat express the neuronal marker, neuron-specific enolase, or the glial marker, glial fibrillary acidic protein, we performed combined immunohistochemistry and autoradiography on brains from adult rats perfused at various times ranging from 1 h to four weeks following [3H]thymidine administration. Light-microscopic examination revealed a negligible number of [3H]thymidine-labeled cells showing neuron-specific enolase immunoreactivity during mitosis. However, by two weeks after [3H]thymidine administration, a significant increase in the density of [3H]thymidine-labeled neuron-specific enolase-immunoreactive cells was detected. Three weeks following [3H]thymidine injection the majority of [3H]thymidine-labeled cells (> 70%) were immunoreactive for the neuronal marker. At the four-week time-point, [3H]thymidine-labeled neuron-specific enolase-immunoreactive cells were indistinguishable from neighboring granule cells. In contrast, glial fibrillary acidic protein immunoreactivity was observed in a small but significant number of [3H]thymidine cells at the 1-h time-point and the proportion of labeled cells that were immunoreactive for this cell marker did not increase with time. [3H]Thymidine-labeled cells that were immunoreactive for glial fibrillary acidic protein typically showed morphologic characteristics of radial glia at all time-points. At the 1-h time-point, the majority of [3H]thymidine-labeled cells were observed in the hilus (> 60%) with the remainder being located in the granule cell layer. However, with a four-week survival-time most [3H]thymidine-labeled cells (> 85%) were located in the granule cell layer. The majority of newly born cells in the adult dentate gyrus differentiate into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)