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.

Introduction

5-Bromo-2-deoxyuridine (BrdU) has been a principal marker for mitotic cells in studies of adult neurogenesis (Gratzner, 1982). The method consists of a pulse injection of BrdU into the intraperitoneal cavity followed by a variable survival time allowing for tracking the fate of divided cells and their progeny. At the end of the experiment, the animal is sacrificed and the tissues fixed with a standard paraformaldehyde-based fixative. BrdU is detected in the tissue using specific primary antibodies. The primary antibodies are then labeled with a secondary antibody tagged with a fluorescent compound or with a substrate for diaminobenzidine (DAB) (Seki and Arai, 1993, Scott et al., 2000). The substitution of an endogenous DNA base, Thymidine, with the BrdU analogue ensures specific labeling of only the dividing cells. This specificity of BrdU combined with the high sensitivity of fluorescence microscopy, including confocal microscopy has led to the great popularity of the method. It is believed that the application of this BrdU method contributed to the current acceptance of neurogenesis as a natural process in the brain of adult mammals (Gross, 2000). One useful feature of BrdU is its long-term retention in divided cells and its passage to their daughter cells. This feature can be used to trace the cell lineage and cell survival. A spectacular example of such use was a study by Eriksson and colleagues who injected patients with BrdU and then examined brain tissue postmortem up to 2 years later. BrdU was detected in the brains of these patients and double labeling confirmed that the surviving cells were neurons (Eriksson et al., 1998).

However, the undeniable usefulness of the BrdU method goes hand in hand with its limitations and drawbacks. The possibility of BrdU producing mutated cells and the consequent severe abnormalities of the developing tissues has been reported (Kolb et al., 1999). These side effects aside, the use of BrdU in whole-animal experiments is difficult due to uncertainty of diffusion of this substance among the various tissues of the body following intraperitoneal injection. For example, the blood brain barrier may prevent the BrdU from freely penetrating the brain tissue, especially in old animals (Cameron and McKay, 2001). In certain species of rodents, the effective incorporation of exogenous tritiated thymidine (3HThy) in DNA synthesis in some tissues was lacking (Adelstein et al., 1964). Since both BrdU and 3HThy may use the same transport mechanisms (Cameron and McKay, 2001), a comparative study of mitosis in different tissues and/or species, using BrdU as a mitotic marker, could be difficult. Stress is known to inhibit neurogenesis (Cameron and Gould, 1994, Gould et al., 1997) and therefore the handling and the BrdU injection procedure is likely to have an unintended effect on the rate of neurogenesis.

Given these drawbacks, an alternative method is highly desirable. Here we report on the use of Ki-67, a nuclear protein, expressed in dividing cells for the entire duration of their mitotic process (Scholzen and Gerdes, 2000). Like BrdU, Ki-67 can be detected with immunohistochemistry. Unlike BrdU, Ki-67 is an endogenous marker that does not have any adverse effects on living cells. Although the function of Ki-67 is not known, it is a reliable marker of mitosis because it is expressed, albeit at different levels, during mitosis and its half-life is very short. Moreover, studies reported thus far show that Ki-67 is expressed during mitosis in all mammalian species from rodents to humans (Scholzen and Gerdes, 2000, Endl and Gerdes, 2000). We focused our attention on the use of Ki-67 in the brain tissue, especially in the hippocampal dentate gyrus, because of the current interest in the possible consequences of nerve cell proliferation on cognition and behaviour (Boonstra et al., 2001a). One example of such use of Ki-67 is already available in the study by Tanapat et al. (1999). In that study a difference between the rate of adult neurogenesis, as indicated by BrdU labeling, between male and female rats was found. Yet the numbers of Ki-67 labeled cells were the same in males and females. They concluded that perhaps the slower cell cycle, that would not be detectable by Ki-67, was responsible for the discrepancy although other interpretations are also possible. In the present study we show a comparison of BrdU and Ki-67 labeling in various experiments and combine their use with the neuronal markers neuronal nuclear protein (NeuN) (Mullen et al., 1992), and collapsin response mediator protein-4 (CRMP-4) (Quinn et al., 1999). Our results will clarify the pros and cons of Ki-67 as a mitotic marker and make its use routine and easier to interpret in future studies of adult neurogenesis.

Section snippets

BrdU administration

The thymidine analogue BrdU (Sigma, St. Louis, MO) was administered intraperitoneally in pulse injections of the stock solution. BrdU stock was prepared in phosphate buffered saline (PBS), (pH 7.2, 0.1 M) with 0.1 N NaOH at 20 mg/ml.

Animal groups

In experiment 1 (effects of intraperitoneal injections) the subjects were young adult (35–40 days) male Wistar rats (Charles River). A pulse injection of 300 mg/kg was given to one group of animals (n=3). To control for possible toxic or stress-related effects due

Results

In experiment 1 we compared the expression pattern of Ki-67 and BrdU in tissue sections taken from brains of laboratory rats (Fig. 1). The two markers showed similar location of the labeled cells, primarily at the border of the hilus (core of the dentate gyrus), and the surrounding granule cell layer (GCL). This region is known as the proliferative zone. A few cells were also found in the hilus. Both markers are present in the nuclei of the labeled cells, as expected. This was shown using BrdU

Discussion

Neurogenesis in the adult dentate gyrus has been observed in all mammalian species examined to date, including humans (Eriksson et al., 1998). To establish a functional significance of the new neurons, one needs to determine that the proliferating cells respond to physiological stimuli, become integrated in the neuronal circuitry and are playing a significant role. Since the dentate gyrus of the hippocampus is one of the hot spots of neurogenesis, it is tempting to speculate that the new cells

Acknowledgements

This research was supported by CIHR and the Heart and Stroke Foundation grants to JMW, and NSERC grant to RB. The assistance of Jason Snyder with the irradiation experiments is greatly appreciated.

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